Autotaxin: motility stimulating protein useful in cancer diagnosis and therapy

ABSTRACT

The present invention relates, in general, to autotaxin. In particular, the present invention relates to a DNA segment encoding autotaxin; recombinant DNA molecules containing the DNA segment; cells containing the recombinant DNA molecule; a method of producing autotaxin; antibodies to autotaxin; and identification of functional domains in autotaxin.

This application is a continuation-in-part of application serial no. 08/249,182 filed May 25, 1994, now abandoned, which is a continuation-in-part of application Ser. No. 07/822,043 filed on Jan. 17, 1992, now U.S. Pat. No. 5,449,753.

FIELD OF THE INVENTION

The present invention relates, in general, to a motility stimulating and compositions comprising the same. In particular, the present invention relates to a purified form of the protein and peptides thereof, for example, autotaxin (herein alternative referred to as "ATX"); a DNA segment encoding autotaxin; recombinant DNA molecules containing the DNA segment; cells containing the recombinant DNA molecule; a method of producing autotaxin; antibodies to autotaxin; and methods of cancer diagnosis and therapy using the above referenced protein or peptides thereof and DNA segments.

BACKGROUND OF THE INVENTION

Cell motility plays an important role in embryonic events, adult tissue remodeling, wound healing, angiogenesis, immune defense, and metastasis of tumor cells (Singer, 1986). In normal physiologic processes, motility is tightly regulated. On the other hand, tumor cell motility may be aberrantly regulated or autoregulated. Tumor cells can respond in a motile fashion to a variety of agents. These include host-derived factors such as scatter factor (Rosen, et al., 1989) and growth factors (Kahan, et al., 1987; Stracke, et al.; Tamm, et al., 1989; Wang, et al. 1990; and Jouanneau, et al. 1991), components of the extracellular matrix (McCarthy, et al. 1984), and tumor-secreted or autocrine factors (Liotta, et al. 1988; Ruff, et al. 1985; Atnip, et al. 1987; Ohnishi, et al. 1990; Silletti, et al. 1991; and Watanabe, et al. 1991).

Many types of host-derived soluble factors act in a paracrine fashion to stimulate cell locomotion. Motility-stimulating proteins called "scatter factors" have been identified which are produced by embryonic fibroblasts and by smooth muscle cells (Stoker, et al. 1987). Scatter factors stimulate random and directed motility by epithelial cells, keratinocytes, vascular endothelial cells and carcinoma cells (Stoker, et al. 1987; Rosen, et al. 1990; and Weidner, et al. 1990), but not fibroblasts. In addition, a number of host-secreted growth factors have been demonstrated to stimulate motility in tumor cells, including nerve growth factor (Kahan, et al. 1987) insulin-like growth factor-I (Stracke, et al. 1988), interleukin-6 (Tamm, et al. 1989), interleukin-8 (Wang, et al. 1990), and acidic fibroblast growth factor (Jouanneau, et al. 1991). These paracrine factors may influence "homing" or the directionality of tumor cell motility.

In contrast to these host-derived factors, many types of tumor cells have been found to produce proteins termed "autocrine motility factors" which stimulate motility by the same tumor cells which make the factor (Liotta, et al. 1986). Autocrine motility factors are not specific for a given type of cancer cell but have a wide spectrum of activity on many types of cancer cells (Kohn, et al. 1990), with little effect on normal fibroblasts or leukocytes.

Autocrine motility factors identified to date act through cell-surface receptors (Stracke, et al. 1987; Nabi, et al. 1990; Watanabe, et al. 1991) resulting in pseudopodial protrusion (Guirguis, et al. 1987) leading to both random and directed migration (Liotta, et al. 1986; Atnip, et al. 1987; Ohnishi, et al. 1990).

Prior studies of human A2058 melanoma cells have demonstrated that these cells are a particularly rich source of autocrine motility factors. An autocrine motility factor with a molecular mass of approximately 60 kDa has been previously isolated from the conditioned media of these cells. (Liotta, et al. 1986). Similar tumor cells derived or induced factors with the same molecular weight have subsequently been reported and purified by several investigators (Atnip, et al. 1987; Schnor, et al. 1988; Ohnishi, et al. 1990; Silletti, et al. 1991; Watanabe et al. 1990). Such factors are thought to play a key role in tumor cell invasion.

Most of the motility factors identified to date have not been purified to homogeneity and have not been sequenced. The novel tumor motility factor of the present invention, named herein as autotaxin ("ATX"), has been purified and verified to be a homogeneous sample by two-dimensional gel electrophoresis. The protein of the present invention is unique from any previously identified or purified motility factor. The molecular size of ATX is about 125 kilo Daltons ("kDa") and it has an isoelectric point of approximately 7.7. ATX stimulates both random and directed migration of human A2058 melanoma cells at picomolar concentrations. The activity of the ATX factor is completely sensitive to inhibition by pertussis toxin. No significant homology has been found to exist between the protein of the invention and any mammalian protein including previous factors known to stimulate cell motility.

There is a great clinical need to predict the aggressiveness of a patient's individual tumor, to predict the local recurrence of treated tumors and to identify patients at high risk for development of invasive tumors. The present invention provides a functional marker which is functionally related to the invasive potential of human cancer. The invention further provides an assay for this secreted marker in body fluids, or in tissues. The assay of the invention can be used in the detection, diagnosis, and treatment of human malignancies and other inflammatory, fibrotic, infectious or healing disorders.

SUMMARY OF THE INVENTION

The present invention relates, generally, to a motility stimulating protein and corresponding peptides thereof, and to a DNA segment encoding same. A human cDNA clone encoding a tumor cell motility-stimulating protein, herein referred to as autotaxin or "ATX", reveals that this protein is an ecto/exoenzyme with significant homology to the plasma cell membrane differentiation antigen PC-1. ATX is a 125 kDa glycoprotein, previously isolated from a human melanoma cell line (A2058), which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations.

It is a specific object of the present invention to provide autotaxin and peptide fragments thereof.

It is a further object of the present invention to provide a DNA segment that encodes autotaxin and a recombinant DNA molecule comprising same. It is a further object of the present invention to provide a cell that contains such a recombinant molecule and a method of producing autotaxin using that cell.

Another object of the present invention is the identification of a transmembrane domain of the human liver autotaxin protein and its apparent absence in tumorous forms of autotaxin. The tumorous form of autotaxin appears to be a secreted protein. The present invention relates to utilization of the different sites of localization for diagnosis and prognosis of the stages of tumor progression. Further, the invention relates to treatment methods, designed to advantageously block the secreted form of autotaxin activity while having little effect on the membrane-bound form of autotaxin.

Yet another object of the present invention relates to the identification of a highly variable region within the autotaxin gene, called a "hot spot". The variations in sequence apparently result in mutations, insertions, deletions and premature termination of translation. The present invention relates to manipulating this region so as to alter the activity of the protein. Further, the hot spot can serve as a marker in tumor diagnosis differentiating between different forms of the autotaxin protein.

It is yet another object of the present invention to provide a method of purifying autotaxin.

It is a further object of the present invention to provide cloned DNA segments encoding autotaxin and fragments thereof. The cDNA encoding the entire autotaxin protein contains 3251 base pairs, and has an mRNA size of approximately 3.3 kb. The full-length deduced amino acid sequence of autotaxin comprises a protein of 915 amino acids. Database analysis of the ATX sequence revealed a 45% amino acid identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I phosphodiesterase expressed on the surface of activated B cells and plasma cells. ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate p-nitrophenyl thymidine-5'monophosphate. Autotaxin now defines a novel motility-regulating function for this class of ecto/exo-enzymes.

Further objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Fractionation of ATX by hydrophobic interaction. A 200 ml sample of A2058 conditioned media was chromatographed on a 200 mL column of phenyl Sepharose-4B. Buffer A was 50 mM Tris (pH 7.5), 5% methanol, and 1.2M ammonium sulfate. Buffer B was 50 mM Tris (pH 7.5), 5% methanol and 50% ethylene glycol. The gradient (----) represents a double linear gradient with decreasing ammonium sulfate (1.2 to 0.0M) and increasing ethylene glycol (0 to 50%). Absorbance was monitored at 280 nm(--) and indicated that most of the protein did not bind to the column. Ten ml fractions were assayed for motility stimulating capacity using the Boyden Chamber assay (∘). The peak of motility activity occurred between 900 and 1050 minutes, ˜12% of the gradient.

FIG. 2. Isolation of ATX by lectin affinity chromatography. 20 ml portions of the phenyl Sepharose activity peak were affinity purified on a 40 ml Concanavalin A Affi-Gel column. The bound components were eluted with a step gradient (----) of methyl α-D-mannopyranoside (0.0 mM, 10 mM, and 500 mM) in a buffer consisting of 0.05M Tris (pH 7.5), 0.1M NaCl, 0.01M CaCl₂ and 20% ethylene glycol. Absorbance was monitored at 280 nm ( ) and indicated that the majority of the protein components did not bind to the column. Motility was assayed in 10 mL fractions (...∘...) and was found predominantly in the 500 mM elution concentration. One of seven chromatographic runs is shown.

FIG. 3. Purification of ATX by weak anionic exchange chromatography. Approximately 30% of the activity peak eluted from the Con A affinity column was applied to a ZORBAX BioSeries-WAX column. The bound components were eluted with an NaCl gradient (----) in a buffer consisting of 10 mM Tris (pH 7.5) and 30% ethylene glycol. Motility (∘) was assayed in 1.0 ml fractions. The peak of activity eluted in a discrete but broad region in the shallow portion of the gradient. Absorbance was monitored at 230 nm ( ). The majority of the protein components not associated with activity bound strongly to the column were eluted at 1.0M NaCl. One of two chromatographic runs is shown.

FIG. 4. Purification of ATX by molecular sieve exclusion chromatography. The entire activity peak eluted from the weak anion exchange column was applied to a series of TSK columns (4000SW, 4000SW, 3000SW, and 2000SW, in this order). Proteins were eluted in a buffer consisting of 0.1M NaPO₄ (pH 7.2) with 10% methanol and 10% ethylene glycol. Two major protein peaks were evident by monitoring the absorbance at 235 nm ( ). Motility (...∘...) was assayed in 0.4 ml samples and found predominantly in the first, smaller, protein peak.

FIG. 5. Final purification of ATX by strong anionic exchange chromatography. Approximately 15% of the activity peak from the molecular sieve exclusion series was applied to a Pro-Pac PA1 column. Protein which bound to the column was eluted with a NaCl gradient (----) in a buffer consisting of 10 mM Tris (pH 7.5), 5% methanol and 20% ethylene glycol. Absorbance was monitored at 215 nM ( ). Motility activity was assayed in 1.0 ml fractions at two different dilutions: 1/5 (...∘....) or 1/15 (. .∘. .). Activity was found to correspond to a double protein peak in the central region of the gradient.

FIG. 6A, 6B and 6C. Protein components associated with the activity peaks from various stages of purification. The activity peak from each chromatographic fractionation was pooled, concentrated and analyzed by SDS-polyacrylamide gel electrophoresis. Molecular weight standards are in Lane 1 for each panel. Panel 6A) 8-16% gradient gel of the first three purification steps, run under non-reducing conditions. Lane 2 is an aliquot of the pooled activity peak eluted from the phenyl sepharose fractionation. Lane 3 is an aliquot of the pooled activity peak eluted from the Con A affinity purification. Lanes 4 and 5 show the "peak" and "shoulder" of activity fractionated by weak anion exchange chromatography (FIG. 3). Panel 6B) 7% gel of the activity peak fractionated by molecular sieve exclusion chromatography. Lanes 2 and 3 show the protein separation pattern of the total pooled activity peak when the gel was run under non-reducing and reducing conditions, respectively. Panel 6C) 8-16% gradient gel of the final strong anionic exchange chromatographic separation, run under non-reducing conditions. Lane 2 comprises ˜1% of the total pooled activity peak eluted from the column.

FIG. 7. Two-dimensional gel electrophoresis of ATX. Purified ATX (FIG. 6, Panel C) was subjected to non-equilibrium isoelectric focusing (5 hr. at 500 v), then applied to a 7.5% SDS-polyacrylamide gel for the second dimension. The pH separation which resulted was measured in 0.5 cm samples of concurrently run tube gels and is shown at the top. Molecular weight standards for the second dimension are shown on the right. This analysis reveals a single component with pI=7.7±0.2 and M_(r) =120,000.

FIG. 8. Dilution curve of ATX. Purified ATX (FIG. 6, Panel C) was serially diluted and tested for motility-stimulating activity. The result, with unstimulated background motility subtracted out, shows that activity is half-maximal at ˜500 pM ATX.

FIG. 9. Pertussis toxin (PT) sensitivity of ATX. A2058 cells were pre-treated for 1 hr. prior to the start of the motility assay with 0.5 μg/ml PT in 0.1% BSA-DMEM or with 0.1% BSA-DMEM alone (for untreated control). The motility activity stimulated by purified ATX (FIG. 6, Panel C) was then assessed for the two treatment groups. The result, expressed as cells/HPF±S.E.M. with unstimulated background motility subtracted out, reveals profound inhibition of PT-treated cells (hatched) compared to untreated cells (solid). PT had no effect on cell viability. S.E.M.'s were <10%.

FIG. 10. Checkerboard analysis of ATX-stimulated motility. Varying dilutions of autotaxin were added to the upper chamber with the cells and/or to the lower chamber, as shown. Motility response, expressed as cells/HPF±S.E.M., was assessed for each point in the checkerboard.

FIG. 11. Purification of ATX peptides on HPLC. ATX, purified to homogeneity by strong anionic exchange chromatography, was sequentially digested by cyanogen bromide, subjected to reduction and pyridylethylation, and digested by trypsin. The resulting peptides were purified on an Aquapore RP300 C-8 reverse phase column using a (0-70) % acetonitrile gradient in 0.1% trifluoroacetic acid (----). The absorbance was monitored at 215 nm ( ) and peaks were collected. Seven peaks, chosen at random for N-terminal amino acid sequence analysis, are shown with appropriate numbers.

FIG. 12. Cloning Strategy, schematically depicted.

FIG. 13. Schematic Diagram of autotaxin gene region.

For A2058: 4C11 is the original DNA clone obtained by screening an A2058 cDNA expression library in λgt11 with anti-peptide ATX-102. Upstream ATX peptide sequences were utilized for PCR amplification of A2058 mRNA, using the technique of reverse transcription/PRC. These peptides include ATX-101, ATX-103, and ATX-224. The approximate localization of each of peptide was obtained by matching the peptide with its homologous region on PC-1.

For N-tera 2D1, a λgt1 0 cDNA library was amplified and the cDNA inserts were isolated. PCR amplification, based on homologies with A2058 sequence, was utilized for DNA sequencing.

For normal human liver, a mRNA from liver was amplified with 5' RACE using primers from the known ATX-224 region of A2058 and N-tera 2D1.

FIG. 14. Schematic match-up of ATX peptides with putative A2058 protein sequence.

FIG. 15. Schematic match-up of ATX peptides with putative N-tera 2D1 protein sequence.

FIG. 16: ATX Treatment with PGNase F. Partially purified ATX was treated with 60 mU/ml PNGase F at 37° C. for 16 hr under increasingly denaturing conditions. The treated ATX samples were separated by SDS polyacrylamide gel electrophoresis run under reducing conditions and stained with Coomassie blue G-250. Lane 1 contains untreated ATX (arrow) with no enzyme added. Lane 2 contains the reaction mixture run under non-denaturing conditions (50 mM tris/10% ethylene glycol, pH 7). Lanes 3 and 4 have added 0.1M β-mercaptoethanol and 0.5% Nonidet-P40, respectively. Lanes 5 and 6 contain the reaction mixtures in which the ATX sample was first boiled for 3 min in 0.1% SDS with (lane 6) or without (lane 5) 0.1M β-mercaptoethanol, then had 0.5% Nonidet-P40 added to prevent enzyme denaturation. The enzyme can be detected as an ˜44 kDa band in lanes 2-6.

FIG. 17: Effect of varying concentrations of PNGase F on ATX molecular weight and motility-stimulating activity. Partially purified ATX was treated with various concentrations (range 0-60 mU/ml, shown on horizontal axis) of PNGase F at 37° C. for 16 hr. FIG. 17A shows the effect of the different treatments on ATX molecular weight. At concentrations of enzyme ≧30 mU/ml, the deglycosylation reaction appears to be complete. FIG. 17B shows the effect of the identical reaction mixtures on motility-stimulating capacity (immediately below the corresponding protein band of FIG. 17A). There is no significant difference between any of the treatment groups.

FIG. 18: Comparison of amino acid sequences of ATX and PC-1. The amino acid sequences of ATX and PC-1 are compared. Amino acid identity is indicated by a vertical line between the sequences. The location of the putative transmembrane/signal sequence is shown by a solid line. The two somatomedin B domains are identified by dashed lines. The putative phosphodiesterase active site is indicated by emboldened lines. The loop region of a single EF hand loop region is identified with double lines. The presumed cleavage site for each protein is indicated with arrows.

FIG. 19: Domain structure of ATX and PC-1. Putative domains are indicated for the two homologous proteins, ATX and PC-1.

DETAILED DESCRIPTION OF THE INVENTION

Tumor cell motility is a critical component of invasion and metatasis, but the regulation of this motility is still poorly understood. At least some tumor cells secrete autocrine motility factors (AMF's) that stimulate motility in the producing cells. Like the analogous autocrine growth factors, these AMF's allow tumor cells independence from the host in this important component of the metastatic cascade. One AMF, autotaxin (ATX), has recently been purified to homogeneity from the human melanoma cell line, A2058 (Stracke, et al., 1992). The purified protein was enzymatically digested and the peptide fragments were separated by reverse phase HPLC. A number of these peptides have been sequenced by standard Edman degradation (Table 6) from different purifications and different enzymatic digestion. Sequence information, obtained initially on 19 purified tryptic peptides, confirmed that the protein is unique with no significant homology to growth factors or previously described motility factors. These peptide sequences have now been used as the basis for identifying and sequencing the cDNA clone for ATX. The present invention comprises an amino acid sequence of ATX as well as a nucleic acid sequence coding for the ATX protein.

                  TABLE 6                                                          ______________________________________                                         PEPTIDE SEQUENCES FOR AUTOTAXIN.                                               PEPTIDE  AMINO ACID                                                            NO.      SEQUENCE            SEQ ID NO:                                        ______________________________________                                         ATX-18   WHVAR               SEQ ID NO: 1                                      ATX-19   PLDVYK              SEQ ID NO: 2                                      ATX-20   YPAFK               SEQ ID NO: 3                                      ATX-29   PEEVTRPNYL          SEQ ID NO: 5                                      ATX-34B  RVWNYFQR            SEQ ID NO: 38                                     ATX-41   HLLYGRPAVLY         SEQ ID NO: 29                                     ATX-48   VPPFENIELY          SEQ ID NO: 7                                      ATX-59   TFPNLYTFATGLY       SEQ ID NO: 32                                     ATX-100  GGQPLWITATK         SEQ ID NO: 8                                      ATX-101/223A                                                                            VNSMQTVFVGYGPTFK    SEQ ID NO: 9                                      ATX-102  DIEHLTSLDFFR        SEQ ID NO: 10                                     ATX-103  TEFLSNYLTNVDDITLVPETLGR                                                                            SEQ ID NO: 11                                     ATX-104  VNVISGPIDDYDYDGLHDTEDK                                                                             SEQ ID NO: 33                                     ATX-204  MHTARVRD            SEQ ID NO: 39                                     ATX-205  FSNNAKYD            SEQ ID NO: 40                                     ATX-209  VMPNIEK             SEQ ID NO: 41                                     ATX-210  TARGWECT            SEQ ID NO: 42                                     ATX-212  (N)DSPWT(N)ISGS     SEQ ID NO: 43                                     ATX-214  LRSCGTHSPYM         SEQ ID NO: 44                                     ATX-215/34A                                                                             TYLHTYES            SEQ ID NO: 45                                     ATX-213/217A                                                                            AIIANLTCKKPDQ       SEQ ID NO: 46                                     ATX-216  IVGQLMDG            SEQ ID NO: 47                                     ATX-218/44                                                                              TSRSYPEIL           SEQ ID NO: 48                                     ATX-223B/24                                                                             QAEVSSVPD           SEQ ID NO: 49                                     ATX-224  RCFELQEAGPPDDC      SEQ ID NO: 50                                     ATX-229  SYTSCCHDFDEL        SEQ ID NO: 51                                     ATX-244/53                                                                              QMSYGFLFPPYLSSSP    SEQ ID NO: 52                                     ______________________________________                                    

ATX is a glycosylated protein due to its high affinity for concanavalin A and amino acid sequence analysis of the ATX peptides. ATX has been demonstrated to be a 125 kDa glycoprotein whose molecular weight reduced to 100-105 kDa after deglycosylation with N-glycosidase F. The calculated molecular weight of the cloned protein is 100 kDa (secreted form) or 105 kDa (full length protein). Based on amino acid composition, the estimated pI is 9.0 which is higher than the pI determined by 2-D gel electrophoresis analysis (7.7-8.0) of purified ATX. This difference can be explained by the presence of sialic acid residues on the sugar moieties.

Autotaxin is secreted by A2058 human melanoma cells cultured in low abundance in serum-free conditioned medium. Autotaxin is a potent new cytokine with molecular mass 125 kDa which has been purified to homogeneity from the conditioned medium of the human melanoma cell line, A2058, utilizing sequential chromatographic methods as described herein. This new cytokine, termed autotaxin (ATX), is a basic glycoprotein with pI˜7.8. ATX is active in the high picomolar to low nanomolar range, stimulating both chemotactic and chemokinetic responses in the ATX-producing A2058 cells as well as other tumor cells. This motile response is abolished by pretreatment of the cells with pertussis toxin. ATX may therefore act through a G protein-linked cell surface receptor. These characteristics distinguish ATX from several small growth factors and interleukins which are implicated in tumor cell motility (Stracke et al., 1988; Ruff et al., 1985; Maciag et al., 1984; Gospodarowicz, 1984; Van Snick, 1990; Yoshimura 1987).

The protein of the present invention, which in one embodiment is derived from A2058 human melanoma cells, can be prepared substantially free from proteins with which it is normally associated using, for example, the purification protocol disclosed herein. Alternatively, the protein of the present invention can be prepared substantially free from proteins, by cloning and expressing the cDNA encoding autotaxin as disclosed herein.

A large volume of serum-free conditioned medium from appropriate producer cells (e.g., tumor cells) is collected and concentrated approximately 500-fold. This concentrated conditioned medium is then separated from other contaminating proteins by techniques that rely on the chemical and physical characteristics of the protein. These include the molecular weight, relative hydrophobicity, net charge, isoelectric focusing point, and the presence of lectin-binding sugar residues on the protein.

Alternatively, the protein, or functional portion thereof, can be synthesized using chemical or recombinant means.

The protein of the present invention has a potent biological activity. Purified ATX is active in the picomolar range and 1 unit of activity corresponds to a concentration of approximately 500 pM as assessed by the cell motility assay described herein and elsewhere (Stracke et al., 1989).

The protein of the present invention has a molecular size, as determined by two dimensional gel electrophoresis, of from 120 to 130 kDa, or more specifically, about 125 kDa. Further, the protein of the present invention can have a pI in the range of 7.5 to 8.0, preferably, approximately 7.7. The present invention relates to autotaxin and peptides thereof having cell motility properties as described herein. These proteins and peptides thereof can be produced by isolation from a natural host or isolation as an expression product from a recombinant host.

The present invention also relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to ATX, or a unique portion of such a sequence (unique portion being defined herein as at least 5, 10, 25, or 50 amino acids). In one embodiment, the DNA segment encodes any one of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:11 and SEQ ID NO:26 to SEQ ID NO:33. Another embodiment uses larger DNA fragments encoding amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:36 and SEQ ID NO:38. The entire coding region for autotaxin can also be used in the present invention shown in SEQ ID NO:66 through SEQ ID NO:69.

In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector (for example plasmid or viral vector) and a DNA segment coding for a polypeptide corresponding to ATX, as can be prepared by one skilled in the art. Preferably, the coding segment is present in the vector operably linked to a promoter. The present invention also relates to a recombinant protein produced from a host cell expressing a cDNA containing a coding region of ATX. Examples of ATX cDNAs from a variety of sources have been cloned and can be used for expression, including inter alia A2058 carcinoma cells, N-tera 2D1 cells and human liver.

In a further embodiment, the present invention relates to a cell containing the above-described recombinant DNA molecule. Suitable host cells include procaryotic cells (such as bacteria, including E. coli) and both lower eucaryotic cells (for example, yeast) and higher eucaryotic cells (for example, mammalian cells). Introduction of the recombinant molecule into the host cells can be effected using methods known in the art.

In another embodiment, the present invention relates to a method of producing a polypeptide having an amino acid sequence corresponding to ATX. The method comprises culturing the above-described cells under conditions such that the DNA segment is expressed, and isolating ATX thereby produced.

In a further embodiment, the present invention relates to an antibody having affinity for ATX or peptide fragments thereof. The invention also relates to binding fragments of such antibodies. In one preferred embodiment, the antibodies are specific for ATX peptides having an amino acid sequence set forth in one of SEQ ID NO:1 through SEQ ID NO:11 and SEQ ID NO:26 through SEQ ID NO:34, SEQ ID NO:36 and SEQ ID NO:38 through SEQ ID NO:52. In addition, the antibodies may recognize an entire autotaxin protein.

Antibodies can be raised to autotaxin or its fragment peptides, either naturally-occurring or recombinantly produced, using methods known in the art.

The ATX protein and peptide fragments thereof described above can be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as carrier proteins. In particular, ATX fragments can be fused or covalently linked to a variety of carrier proteins, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See for example, Harper and Row, (1969); Landsteiner, (1962); and Williams et al., (1967), for descriptions of methods of preparing polyclonal antisera. A typical method involves hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts. Description of techniques for preparing such monoclonal antibodies may be found in Stites et al., and references cited therein, and in particular in Kohler and Milstein (1975), which discusses one method of generating monoclonal antibodies.

In another embodiment, the present invention relates to an oligonucleotide probe synthesized according to the sense or antisense degenerative sequence set forth in one of SEQ ID NO:1 through SEQ ID NO:11, SEQ ID NO:26 through SEQ ID NO:33, SEQ ID NO:39 through SEQ ID NO:52, and SEQ ID NO:55 through SEQ ID NO:65.

Protein database searches of this sequence revealed a 45% amino acid identity with the plasma cell membrane marker protein, PC-1. ATX and PC-1 appear to share a number of domains, including two somatomedin B domains, the loop region of an EF hand, and the enzymatic site of type I phosphodiesterase/nucleotide pyrophosphatase. Like PC-1, ATX hydrolyzes p-nitrophenyl thymidine-5'-monophosphate, a type 1 phosphodiesterase substrate. This enzymatic function of ATX suggests a newly identified function for ecto/exo-enzymes in cellular motility.

In a further embodiment, the present invention relates to a method of diagnosing cancer metastasis and to a kit suitable for use in such a method. Preferably, antibodies to ATX can be used in, but not limited to, ELISA, RIA or immunoblots configurations to detect the presence of ATX in body fluids of patients (e.g. serum, urine, pleural effusions, etc.). These antibodies can also be used in immunostains of patient samples to detect the presence of ATX.

In yet another embodiment, the present invention relates to in vivo and in vitro diagnostics. ATX may be radiolabelled, by means known to one skilled in the art, and injected in cancer patients with appropriate ancillary substances also known to one skilled in the art, in order to ultimately detect distant metastatic sites by appropriate imagery. The level of ATX in tissue or body fluids can be used to predict disease outcomes and/or choice of therapy which may also include ATX inhibitors.

In a further embodiment, the present invention relates to a treatment of cancer. ATX antibodies can be cross-linked to toxins (e.g., Ricin A), by means known to one skilled in the art, wherein the cross-linked complex is administered to cancer patients with appropriate ancillary agents by means known to one skilled in the art, so that when the antibody complex binds to the cancer cell, the cell is killed by the cross-linked toxin.

In another embodiment, the different localizations of the normal versus tumorous forms of the ATX proteins within the tissue can be used as a tool for diagnosis and prognosis. The stage of disease progression can be monitored by elevated levels of ATX in the extracellular space as opposed to its normal cell membranes association. In addition, treatment methods for control of tumor progression can be designed to specifically block the activity of the secreted form of ATX. Such methods would have a preferential effect upon secreted ATX during tumor progression while not effecting normal ATX formation.

Yet another embodiment utilizes the hot spot located in the region from approximately nucleotides 1670 through 1815, as a marker gene for identification of tissues carrying a tumorous form of ATX.

The present invention is described in further detail in the following non-limiting examples.

EXAMPLES

The following protocols and experimental details are referenced in the Examples that follow:

Materials.

The polycarbonate Nuclepore membranes and the 48-well microchemotaxis chambers were obtained from Neuro Probe, Inc. Pertussis toxin (PT), ethylene glycol (biotechnology grade), methyl α-D-mannopyranoside were obtained from commercial vendors. The ampholyte, pH 3-10 Bio-Lyte and pH 8-10 Bio-Lyte, were obtained from Bio-Rad. Phenyl Sepharose CL-4B; affi-Gel concanavalin A; ZORBAX BioSeries-WAX (weak anion exchange) column (9.4 mm×24 cm); Spherogel-TSK 4000SW, 3000SW and 2000SW columns (each 7.5 mm×30 cm); the Pro-Pac PA1 (4×50 mm) strong anion exchange column; the Aquapore RP300 C-8 reverse phase column (220×2.1 mm); and the AminoQuant C-18 reverse phase column (200×2.1 mm) were also obtained from commercial sources.

Affi-Gel 10 affinity resin was from Bio-Rad. The GeneAmp PCR Reagent kit with AmpliTaq and the GeneAmp RNA PCR kit were purchased from Perkin-Elmer. The 5' RACE kit came from Gibco BRL Life Technologies, Inc. The p-nitrophenyl thymidine-5'monophosphate was obtained from Calbiochem Biochemicals.

Ethylene glycol (biotechnology grade) was from Fisher Biochemicals (Pittsburg, Pa). Peptide N-glycosidase F ("PNGase F"), O-glycosidase, neuraminidase (Arthrobacter ureafaciens), and swainsonine ("Swn") came from Boehringer-Mannheim (Indianapolis, Ind.). 1-Deoxymannojirimycin ("dMAN"), and N-methyl-1-deoxynojirimycin ("NMdNM") were from Oxford GlycoSystems, Inc. (Rosedale, N.Y.). Biotinylated concanavalin A, HRP-conjugated streptavidin, and HRP-conjugated goat anti-rabbit immunoglobulin were purchased from Pierce Chemicals (Rockford, Ill.). Polyvinyl pyrrolidone-free polycarbonate membranes and the microchemotaxis chamber were from NeuroProbe, Inc. (Cabin John, Md.).

Cell Culture.

The human melanoma cell line A2058, originally isolated by Todaro (Todaro et al., 1980), was maintained as previously described by Liotta (Liotta et al., 1986). The N-tera 2 (D1 clone) was a kind gift from Dr. Maxine Singer, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health and was maintained as described (Andrews, P. W., Goodfellow, P. N. and Bronson, D. L. (1983) Cell surface characteristics and other markers of differentiation of human teratocarcinoma cells in culture.).

Production of Autotaxin.

A2058 cells were grown up in T-150 flasks, trypsinized, and seeded into 24,000 cm² cell factories at a cell density of 1×10¹⁰ cells/factory. After 5-6 days, the serum-containing medium was removed and the cells were washed with DPBS. The factories were maintained in DMEM without phenol red, supplemented with 4 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml crystallized bovine serum albumin, 10 μg/ml bovine insulin, and 1 μM aprotinin. Culture supernatants were harvested every 3 days, frozen at -40° C. and replaced with fresh serum-free medium. Each cycle of supernatant was tested for ATX production with a cell motility assay detailed below. Typically, a cell factory continued to be productive for 9-11 of these cycles.

After accumulation of approximately 45-60 L of supernatant, the culture supernatants were thawed and concentrated down to 2-2.5 L using an Amicon S10Y30 spiral membrane ultrafiltration cartridge. This supernatant was further concentrated in an Amicon high performance ultrafiltration cell using Diaflo membranes. The final volume achieved from 100-200 L of conditioned medium was typically 250-400 ml. All ultrafiltrations were performed at 4° C.

Cell Motility Assays.

Purification of autotaxin was monitored by testing the motility-stimulating capacity of the fractions collected from the columns. These fractions were in buffers unsuitable for a chemotaxis assay so each fraction had to be washed into an appropriate buffer, i.e., 0.1% (w/v) BSA in DPBS containing calcium and magnesium. This dialysis was performed by adding aliquots of each fraction to be tested into Centricon-30™ ultrafiltration tubes, which retain molecular species larger than 30,000 daltons.

The assay to determine motility was performed in triplicate using a 48-well microchemotaxis chamber as described elsewhere in detail (Stracke et al., 1987; Stracke, et al., 1989). The Nuclepore™ membranes used in these modified Boyden chambers were fixed and stained with Diff-Quik.™ Chemotaxis was quantitated either by reading the stained membranes with a 2202 Ultroscan laser densitometer or by counting 5 randomly chosen high power fields (HPF) under light microscopy (400×) for each replicate. Densitometer units (wavelength--633 nm) have been shown to be linearly related to the number of cells per HPF (Taraboletti, 1987; Stracke, et al., 1989). Typically, unstimulated motility (background) corresponded to 5-10 cells/HPF and highly responding cells to 70-100 cells/HPF above unstimulated background (i.e., 75-110 total cells/HPF).

For experiments using PT, the toxin was pre-incubated with the cells for 1-2 hr. at room temperature prior to the assay and maintained with the cells throughout the assay (Stracke, et al., 1987). The treated cells were tested for their motility response to the chemoattractant as well as for unstimulated random motility.

Purification of Autotaxin.

Ammonium sulfate, to a final concentration of 1.2M, was added to the concentrated A2058 conditioned medium for 1 hr. at 4° C. The solution was spun in a RC2-B Ultraspeed Sorvall centrifuge at 10,000×g for 15 min. Only the supernatant had the capacity to stimulate motility.

In the first step, the sample was fractionated by hydrophobic interaction chromatography using 200 ml phenyl Sepharose CL-4B column equilibrated into 50 mM Tris (pH 7.5), 5% (v/v) methanol and 1.2M ammonium sulfate. The supernatant from the ammonium sulfate fractionation was added to this column and eluted using linear gradients of 50 mM Tris (pH 7.5), 5% (v/v) methanol, with decreasing (1.2-0.0) M ammonium sulfate and increasing (0-50) % (v/v) ethylene glycol at 1 ml/min.

The active peak was pooled, dialyzed into 50 mM Tris, 0.1M NaCl, 0.01M CaCl₂, 20% (v/v) ethylene glycol, and subjected to a second fractionation by lectin affinity chromatography using a 40 ml Affi-Gel concanavalin A column run at 1 ml/min. The sample was eluted in a stepwise fashion in the same buffer with 0, 10, and 500 mM methyl α-mannopyranoside added successively. Fractions from each step of the gradient were pooled and tested for their capacity to stimulate motility.

In the third purification step, the sample that eluted at 500 mM α-methyl-mannopyranoside was dialyzed into 10 mM Tris (pH 7.5) with 30% (v/v) ethylene glycol and fractionated by weak anion exchange chromatography. Chromatography was carried out on a ZORBAX BioSeries-WAX column using a Shimadzu BioLiquid chromatograph and eluted with a linear gradient of (0.0-0.4M) sodium chloride at 3 ml/min.

The active peak was pooled, dialyzed against 0.1M sodium phosphate (pH 7.2), 10% (v/v) methanol, and 10% (v/v) ethylene glycol, and subjected to a fourth fractionation step on a series of Spherigel TSK columns (4000SW, 4000SW, 3000SW, 2000SW, in that order). This molecular sieve step was run using the Shimadzu BioLiquid chromatograph at 0.4 ml/min.

The active peak was pooled and dialyzed into 10 mM Tris (pH 7.5), 5% (v/v) methanol, 20% (v/v) ethylene glycol and subjected to a fifth (strong anion exchange) chromatography step, a Pro-Pac PA1 column run at 1 ml/min using a Dionex BioLC with AI450 software. The sample was eluted with a linear gradient of (0.0-0.4M) NaCl.

In order to calculate activity yields after each step of purification, a unit of activity had to be derived. The dilution curve of ATX was biphasic with a broad peak and a linear range at sub-optimal concentrations. One unit of activity/well (i.e., 40 units/ml) was defined as 50% of the maximal activity in a full dilution curve. This allowed calculation of the activity contained in any volume from the dilution needed to achieve 1 unit/well. Therefore, if a 1:10 dilution were needed in order to produce 1 unit of activity/well, the material contained 10×40=400 units/ml.

Gel Electrophoresis.

Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis using the conditions of Laemmli (Laemmli, 1970). In brief, 7 or 8% SDS-containing polyacrylamide gels were prepared or pre-poured (8-16%) gradient gels were obtained commercially. Samples were prepared with or without reducing conditions (5% β-mercaptoethanol). After electrophoretic separation, the gels were stained using Coomassie Blue G-250 as previously described (Neuhoff, et al., 1988). In this staining protocol, which ordinarily requires no destaining step, the Coomassie stain appears to be able to stain as little as 10 ng of protein.

For two-dimensional electrophoresis, the protein, in 20% ethylene glycol, was dried in a Speed-vac and redissolved in loading solution: 9M urea, 1% (v/v) pH 3-10 Bio-Lyte, and 2.5% (v/v) Nonidet-P40. This sample was then subjected to isoelectric focusing (O'Farrell, 1975) using a Bio-Rad tube cell in 120×3 mm polyacrylamide tube gels containing 9M urea, 2% (v/v) pH 3-10 Bio-Lyte, 0.25% (v/v) pH 8-10 Bio-Lyte and 2.5% (v/v) Nonidet-P40. Reservoir solutions were 0.01M phosphoric acid and 0.02M NaOH. Non-equilibrium isoelectric focusing (O'Farrell, et al., 1977) was run initially with constant voltage (500 v) for 5 hr. Since the protein was basic, the procedure was repeated under equilibrium conditions (500 v for 17 hr.). Electrophoresis in the second dimension was performed on a 7.5% polyacrylamide using the conditions of Laemmli (1970). The gel was stained with Coomassie Blue G-250 as above.

Preparation of peptides for internal sequence of autotaxin.

Homogeneous ATX was sequentially digested with cyanogen bromide and, following reduction and pyridylethylation, with trypsin (Stone, et al., 1989). The resulting fragments were then separated by gradient elution on an Aquapore RP300 C-8 reverse phase column: 0.1% (v/v) trifluoroacetic acid and (0-70) % acetonitrile over 85 min. at a flow rate of 0.2 ml/min. A Dionex AI450 BioLC system was utilized and fractions were collected manually while monitoring the absorbance at 215 nm. Sequence analysis of peptides.

The amino acid sequences of peptides resulting from digestion and purification of ATX peptides #1-7 and 12-18, corresponding to SEQ ID NO:1 through SEQ ID NO:7 and SEQ ID NO:26 through SEQ ID NO:32, respectively, were determined on a Porton Instruments 2020 off-line sequenator using standard program #1. Phenylthiohydantoin amino acid analysis of sequenator runs were performed on a Beckman System Gold HPLC using a modified sodium acetate gradient program and a Hewlett-Packard C-18 column. ATX-100 (SEQ ID NO:8), ATX-101 (SEQ ID NO:9), ATX-102 (SEQ ID NO:10), ATX-103 (SEQ ID NO:11) and ATX 104 (SEQ ID NO:33) were sequenced from gel-purified ATX.

Protein databases (Pearson, et al. 1988) that were searched for homologies in amino acid sequence with the ATX peptides include: GenBank (68.0), EMBL (27.0), SWISS-PROT (18.0), and GenPept (64.3).

Example 1 Purification of Autotaxin

The A2058 cells had been previously shown to produce protein factors which stimulate motility in an autocrine fashion (Liotta, et al., 1986). Conditioned medium from these cells was therefore used to identify and purify a new motility-stimulating factor, which is here named autotaxin and referred to as ATX. Since the purification was monitored with a biological assay, motility-stimulating activity had to be maintained throughout. The activity proved to be labile to freezing, acidic buffers, proteases (but not DNase or RNase), reduction, strong chaotrophic agents (e.g. >4M urea), and a variety of organic solvents (isopropanol, ethanol, acetonitrile). An organic solvent, ethylene glycol, which did not decrease bioactivity, was added for both storage and chromatographic separation.

100-200 L of serum-free conditioned medium were required in order to produce enough ATX for amino acid sequence analysis. The medium contained low concentrations of both BSA (5 μg/ml) which was needed as a carrier protein and insulin (10 μg/ml) which was required to support cell growth in low protein medium. Ultrafiltration to concentrate this large volume was performed with low protein-binding YM30 membranes which retain molecular species with M_(r) >30,000. As seen in Table 1, 200 L of conditioned medium prepared in this manner resulted in 10×10⁶ units of activity. However, the initial unfractionated conditioned medium contained additional substances known to stimulate activity, particularly insulin, which does not completely wash out in the ultrafiltration step and which is additive to the motility stimulating activity in a complex manner (Stracke, et al., 1989). This had to be taken into account in order to determine yields for subsequent steps in which insulin had been removed.

                  TABLE 1                                                          ______________________________________                                         PURIFICATION OF AUTOTAXIN                                                                                       Specific                                      Purification Step                                                                            Protein  Activity.sup.a                                                                           Activity                                      Recovery      (mg)     (total units)                                                                            (units/mg)                                                                            (%).sup.b                              ______________________________________                                         200 L Conditioned                                                                            33,000   10,000,000.sup.c                                                                         300                                           Medium                                                                         Phenyl Sepharose                                                                             1,235    460,000   370    100                                    Concanavalin A                                                                               58       660,000   11,400 100                                    Weak Anion Exchange                                                                          4.5      490,000   110,000                                                                               100                                    TSK Molecular Sieves                                                                         ˜0.4.sup.d                                                                        220,000   550,000                                                                               48                                     Strong Anion Exchange                                                                        ˜0.04.sup.d                                                                       24,000.sup.e                                                                             600,000                                                                               5.2                                    ______________________________________                                          .sup.a Activity calculated from Boyden chamber assay. The dilution which       resulted in 50% of maximal activity (generally approximately 20 laser          density units or ˜40 cells/HPF) was chosen to have 1 unit of             activity per well (equivalent to 40 units/ml).                                 .sup.b Recovery was estimated from activity, after the first purification      column (i.e., phenyl sepharose).                                               .sup.c Initial activity in the unfractionated conditioned medium reflecte      the fact that insulin was used in the medium as a necessary growth factor      under low protein conditions.                                                  .sup.d Estimated protein is based on quantificafion by amino acid              analysis.                                                                      .sup.e This specific activity for purified protein corresponds to              ˜10 fmol ATX/unit of motility activity (in a Boyden chamber well). 

The first step in the purification involved fractionation by hydrophobic interaction chromatography using a phenyl Sepharose CL-4B column. The results are shown in FIG. 1. Most proteins, including insulin, eluted from the column in early fractions or in the void. However, the peak of activity eluted relatively late. The activity which was purified was estimated as 460,000 units±20% (Table 1). As the pooled peak of activity from the phenyl Sepharose fractionation is considered to be the first sample without significant insulin contamination, subsequent yields are measured against its total activity. Gel electrophoresis of a small portion of the pooled peak of activity (FIG. 6A, column 2) revealed a large number of protein bands with BSA predominant from the original conditioned medium.

In the second step of purification, the active peak was applied to the lectin affinity column, Affi-Gel concanavalin A. As shown in FIG. 2, most protein (estimated to be 90% of the total absorbance at 280 nm) failed to bind to the column at all. The non-binding fraction contained essentially no motility-stimulating activity (see dotted line in FIG. 2). When a linear gradient of methyl α-D-mannopyranoside was applied to the column, chemotactic activity eluted off in a prolonged zone, beginning at a concentration of approximately 20 mM sugar. Consequently, a step gradient was used to elute. Pure BSA failed to bind to con A.

Activity was found primarily in the 500 mM step of methyl α-D-mannopyranoside. There appeared to be no significant loss of activity as seen in Table 1; however, specific activity (activity/mg total protein) increased thirty-fold. Gel electrophoresis of the pooled and concentrated peak (FIG. 6A, column 3) revealed that the BSA overload was no longer apparent and the number of bands were much reduced. When the unbound protein was concentrated and applied to a gel, it appeared identical to the active peak from phenyl Sepharose-4B with a large BSA band.

The third purification step involved fractionating the previous active peak by weak anion exchange chromatography as shown in FIG. 3. Under the running conditions, activity eluted in a broad peak-shoulder or double peak in the middle of the shallow portion (0.0-0.4M) of the NaCl gradient. The largest proportion of protein, lacking in motility-stimulating capacity, bound strongly to the column and eluted off in high salt (1M NaCl). There appeared to be no significant loss of activity, though specific activity increased by twenty-fold (Table 1). Analysis by gel electrophoresis of both the peak (28-34 min. in FIG. 3) and the shoulder (35-45 min. in FIG. 3) is shown in FIG. 6A (columns 4 and 5, respectively). Two predominant protein bands resulted: a broad doublet around 25-35 kDa and a second doublet around 110-130 kDa.

In the fourth purification step, the active peak was applied to a series of molecular sieves. Spectrophotometric monitoring of the eluant revealed two large peaks of protein (FIG. 4). Activity corresponded to the first, higher molecular weight peak. Recovery of activity was ˜48% with a five-fold increase in specific activity. Analysis by gel electrophoresis was performed under non-reducing and reducing conditions as shown in FIG. 6B (columns 2 and 3, respectively). This fractionation step had essentially removed all contaminating protein of molecular weight <55 kDa. The predominant band remaining has a molecular weight of 120 kDa unreduced and 125 kDa reduced; there are two minor bands with molecular weights 85 kDa and 60 kDa. The fact that the 120 kDa protein changes so little in electrophoretic mobility after reduction would tend to indicate a paucity of disulfide bonds. However, the existing disulfide bonds have functional significance because motility-stimulating activity is labile to reduction.

The fifth purification step involved fractionation of the active peak by strong anion exchange chromatography. As shown in FIG. 5, activity corresponds to two broad optical absorbance peaks in the middle of the gradient with contaminating proteins eluting earlier. These two peaks were identical by amino acid analysis and by polyacrylamide gel electrophoretic separation. They presumably represent different glycosylation states of the same parent protein. Activity is shown in FIG. 5 at two different sample dilutions. Several dilutions of the fractionated samples were often necessary in order to resolve the true "peak" of activity as the shape of the ATX dilution curve was not sharp due to saturation and down regulation at high concentrations. Recovery from this chromatographic step is lower (5% compared to phenyl Sepharose), as might be expected when a minute quantity of protein is applied to a column; however, specific activity again increased (Table 1). Analysis by gel electrophoresis revealed a single protein band at molecular weight 120 kDa, unreduced (FIG. 6C, column 2).

Example 2 Characterization of Autotaxin

Two dimensional gel electrophoresis of the purified protein (FIG. 7) revealed a single predominant band. The band slopes downward slightly toward the basic side of the gel in a manner that is characteristic of glycosylated proteins. A basic pI of 7.7±0.2 was essentially the same whether the isoelectric focusing was run under non-equilibrium conditions (5 hr.) or was allowed to go to equilibrium (17 hr.).

A dilution curve of the purified protein is shown in FIG. 8. The protein is active in the picomolar range and 1 unit of activity appears to correspond to a concentration of 400-600 picomolar (or approximately 10 fmol of ATX/Boyden chamber well). When dilutions were begun at higher concentrations of ATX, the resultant curve showed a broad plateau with down-regulation at the highest concentrations. The motility response to purified autotaxin is highly sensitive to pertussis toxin (hereinafter referred to as "PT") (Table 2 and FIG. 9) with approximately 95% inhibition of activity at 0.5 μg/ml PT.

                  TABLE 2                                                          ______________________________________                                         Effect of Pertussis Toxin (PT) on Autotaxin-stimulated motility                          A2058 Motility Response                                                        (density units.sup.1)                                                          control cells.sup.2                                                                     Pertussis toxin-treated cells.sup.3                         ______________________________________                                         Condition medium.sup.4                                                                     60.3       0.4                                                     Purified Autotaxin                                                                         38.5       0.0                                                     ______________________________________                                          .sup.1 Chemotaxis quantitated by motility assay (Stracke, et al., 1978).       .sup.2 A2058 cell suspended at 2 × 10.sup.6 cells/ml in DMEM             supplemented with 1 mg/ml bovine serum and rocked at room temperature for      1 hr.                                                                          .sup.3 As control with 0.5 μg/ml pertussis toxin.                           .sup.4 Prepared by adding DMEM without phenol red supplemented with 0.1        mg/ml bovine serum albumin to subconfluent flasks of A2058 cells. The          medium was harvested after 2 days incubation at 37° C. in a             humidified atmosphere and concentrated 2530 fold using an Amicon               ultrafiltration assembly with a YM30 membrane                            

Checkerboard analysis was performed to assess the random (chemokinetic) versus the directed (chemotactic) nature of the motility response to ATX. Chambers were assembled with different concentrations of ATX above and below the filter, using ATX purified through the weak anion exchange fractionation step. Squares below the diagonal reflect response to a positive gradient, squares above reflect response to a negative gradient, and squares on the diagonal reflect random motility in the absence of a gradient. ATX stimulates both chemotactic and chemokinetic responses (FIG. 10), with chemotactic responses as high as fifteen-fold above background and chemokinesis as high as eight-fold above background.

Amino acid analysis after complete acid hydrolysis was used to quantitate purified protein. This hydrolysis was carried out on protein excised from a polyacrylamide gel and presumed to be pure. The analysis indicated that 2.7 nmol of protein was present after fractionation on the molecular sieve. After fractionation by strong anion exchange chromatography, approximately 300 pmol remained. The results of the analysis are shown in Table 3.

                  TABLE 3                                                          ______________________________________                                         AMINO ACID COMPOSITION OF AUTOTAXIN                                            (CYS and TRP were not determined in this analysis)                             Amino Acid    Residues/100                                                     ______________________________________                                         ASX           12.5                                                             THR           6.0                                                              SER           5.7                                                              GLX           9.4                                                              PRO           7.4                                                              GLY           7.0                                                              ALA           3.9                                                              VAL           6.7                                                              MET           1.2                                                              ILE           4.3                                                              LEU           9.0                                                              TYR           5.2                                                              PHE           5.2                                                              HIS           3.8                                                              LYS           7.4                                                              ARG           5.4                                                              ______________________________________                                    

Example 3 ATX Degradation and Determination of Amino Acid Sequence

Attempts to obtain N-terminal sequence information from purified ATX repeatedly proved futile. The purified protein was therefore, sequentially digested and the resulting peptides fractionated by reverse phase chromatography. The results are shown in FIG. 11. Multiple sharp peaks including clusters at both the hydrophilic and hydrophobic ends of the gradient are seen.

Several of these peptide peaks were chosen randomly for Edman degradation and N-terminal amino acid sequence analysis. Seven of the eight peaks (shown in FIG. 11) chosen gave clear single sequence information as as seen in Table 4. Using material from a separate digestion and purification, the remaining four sequences were also obtained.

Separate sense and antisense oligonucleotide probes were synthesized according to the fragment sequences of Table 4 by methods known to one skilled in the art. Representative probes are shown in Table 5.

                                      TABLE 4                                      __________________________________________________________________________     Peptide sequences for Autotaxin.                                               PEPTIDE                                                                               AMINO ACID        SEQ ID:                                               NO.    SEQUENCE          NO:     NAME                                          __________________________________________________________________________     1.     WHVA              SEQ ID NO: 1                                                                           ATX 18                                        2.     PLDVYK            SEQ ID NO: 2                                                                           ATX 19                                        3.     YPAFK             SEQ ID NO: 3                                                                           ATX 20                                        4.     QAEVS             SEQ ID NO: 4                                                                           ATX 24                                        5.     PEEVTRPNYL        SEQ ID NO: 5                                                                           ATX 29                                        6.     YDVPWNETI         SEQ ID NO: 6                                                                           ATX 47                                        7.     VPPFENIELY        SEQ ID NO: 7                                                                           ATX 48                                        8.     GGQPLWITATK       SEQ ID NO: 8                                                                           ATX 100                                       9.     VNSMQTVFVGYGPTFK  SEQ ID NO: 9                                                                           ATX 101                                       10.    DIEHLTSLDFFR      SEQ ID NO: 10                                                                          ATX 102                                       11.    TEFLSNYLTNVDDITLVPETLGR                                                                          SEQ ID NO: 11                                                                          ATX 103                                       12.    QYLHQYGSS         SEQ ID NO: 26                                                                          ATX 37                                        13.    VLNYF             SEQ ID NO: 27                                                                          ATX 39                                        14.    YLNAT             SEQ ID NO: 28                                                                          ATX 40                                        15.    HLLYGRPAVLY       SEQ ID NO: 29                                                                          ATX 41                                        16.    SYPEILTPADN       SEQ ID NO: 30                                                                          ATX 44                                        17.    XYGFLFPPYLSSSP    SEQ ID NO: 31                                                                          ATX 53                                        18.    TFPNLYTFATGLY     SEQ ID NO: 32                                                                          ATX 59                                        19.    VNVISGPIFDYDYDGLHDTEDK                                                                           SEQ ID NO: 33                                                                          ATX 104                                       __________________________________________________________________________

Peptide numbers 1-7 refer to peaks numbered in FIG. 11. Peptide numbers 12-18 refer to peptides purified from the preparation which yielded peptide numbers 1-7. Peptides 8-11 and 19, are from a separate purification, not shown in FIG. 11.

X refers to potentially glycosylated residues.

                  TABLE 5                                                          ______________________________________                                         Oligonucleotides synthesized from peptide sequences of autotaxin               (ATX). The number of the oligonucleotide corresponds to the ATX                peptide number as per Table 4. The final letter suffix distinguishes           whether the oligonucleotide is a sense (S) or antisense (A) sequence.          Oligo Sequence               SEQ ID NO:                                        ______________________________________                                         A-18A GTT-GGC-AGC-NAC-RTG-CCA                                                                               SEQ ID NO: 12                                     A-18S TGG-CAY-GTN-GCT-GCC-AAC                                                                               SEQ ID NO: 13                                     A-20A CTT-GAA-GGC-AGG-GTA    SEQ ID NO: 14                                     A-20S TAY-CCT-GCN-TFY-AAG    SEQ ID NO: 15                                     A-29A GGT-NAC-YTC-YTC-AGG    SEQ ID NO: 16                                     A-29S CCT-GAR-GAR-GTN-ACC    SEQ ID NO: 17                                     A-47A NGT-NGC-RTC-RAA-TGG-CAC-RTC                                                                           SEQ ID NO: 18                                     A-47S GAY-GTG-CCA-TTY-GAY-GCN-ACN                                                                           SEQ ID NO: 19                                     A-48A GTT-DAT-RTT-STC-RAA-TGG-GGG                                                                           SEQ ID NO: 20                                     A-48S CCC-CCA-TTT-GAG-AAC-ATC-AAC                                                                           SEQ ID NO: 21                                     A-100A                                                                               CTT-NGT-NGC-NGT-DAT-CCA-NAR-                                                                          SEQ ID NO: 22                                           GGG-YTG-GCC-GCC                                                          A-100S                                                                               GGC-GGC-CAR-CCC-YTN-TGG-ATH-                                                                          SEQ ID NO: 23                                           ACN-GCN-ACN-AAG                                                          A-101A                                                                               CTT-RAA-GGT-GGG-GCC-RTA-GCC-                                                                          SEQ ID NO: 24                                           CAC-RAA-GAC-TGT-YTG-CAT                                                  A-101S                                                                               ATG-CAR-ACA-GTC-TTY-GTG-GGC-                                                                          SEQ ID NO: 25                                           TAY-GGC-CCC-ACC-TTY-AAR                                                  ______________________________________                                    

Example 4 Antipeptide Antibodies

Rabbits were injected with ATX-101 (SEQ ID NO:10) which had been cross-linked to bovine serum albumin. Antisera from these rabbits was subjected to salt precipitation followed by purification using affinity chromatography with Affi-Gel 10 beads covalently linked to the peptide, ATX-101 (SEQ ID NO:10). This affinity purified antibody reacted with the partially purified protein on immunoblots. This same antibody has been used to perform immunohistochemical stains on human tissue.

Example 5 Enzymatic Deglycosylation of ATX

Purified ATX that was to be treated with peptide N-glycosidase F (PNGase F) was first dialyzed into 0.2M sodium phosphate, 10% (v/v) ethylene glycol pH 7.0, using Centricon-30 ultrafiltration tubes. Varying concentrations of PNGase F were added to the ATX and incubated 16-18 hr. at 37° C. Complete digestion appeared to occur at concentrations of enzyme above 30 mU/ml (where 1 U converts 1 mmol of substrate/min). For comparison, the experiments were repeated in the presence of 0.1M β-mercaptoethanol or 0.1% (w/v) SDS plus 0.5% (v.v) Nonidet-P40. ATX that was to be treated with neuraminidase or O-glycosidase was dialyzed into 20 mM sodium phosphate, 0.1M calcium acetate, and 10% (v/v) ethylene glycol (pH 7.2). Neuraminidase was added to a final concentration of 2 U/ml. For treatment with neuraminidase alone, this mixture was incubated 16-18 hr at 37° C. Since O-glycosidase requires the removal of terminal sialic acid residues for efficient deglycosylation, ATX was pre-incubated with neuraminidase for 30-125 mU/ml and incubated 16-18 hr. at 37° C. The treated ATX was then dialyzed into 50 mM Tris with 20% ethylene glycol for storage at 5% C.

Treatment of ATX with N-glycosylation altering agents

A2058 cells were split into four 150 cm² flasks and incubated until just subconfluent in DMEM supplemented with 10% fetal calf serum. The medium was then replaced with fresh 10% FCS/DMEM to which had been added DPBS for control, 1 mM dMAN, 1 mM NMdNM, or 10 mM (1.7 mg/ml) Swn. Concentrations of these pharmacological agents were similar to those previously described as inhibiting N-glycan processing enzymes in melanoma cells (Seftor, et al. 1991; Dennis, et al. 1990) as well as carcinoma cells (Ogier, et al. 1990). On the next day, each flask was washed twice with Dulbecco's phosphate buffered saline with calcium ("DPBS") then 20 ml of Dulbecco's minimum essential medium ("DMEM") supplemented with 0.01% (w/v) bovine serum albumin ("BSA") was added. The same concentration of each agent was added to the appropriate equilibrated flask and incubated for ˜24 hr, after which the medium from each treatment group was collected, concentrated, washed into DPBS and stored at 5° C.

Cells from each flask were trypsinized and counted. There was no loss of viability or reduced cell number in any of the treatment groups compared to control cells.

Effect of PNGase F on ATX

ATX binds to concanavalin A ("Con A") agarose beads and is eluted with buffer containing 0.5M methyl a-D-mannopyranoside, indicating that ATX is likely to contain mannose residues. Such mannose sugar residues are most characteristic of N-linked oligosaccharides. In order to verify that ATX contained asparagine-linked oligosaccharides, we treated it with the endoglycosidase, PNGase F, which cleaves high mannose, hybrid, and complex N-linked oligosaccharides at the asparagine residue.

Partially purified ATX was treated with 60 mU/ml of enzyme under a variety of increasingly denaturing conditions and then separated by polyacrylamide gel electrophoresis (FIG. 16). Lane 1 shows untreated material; the 125 kDA band (arrow) is autotaxin. When this material is treated overnight with PNGase F under very mild conditions, the size of the 125 kDa band decreases to ˜100-105 kDa. Addition of 0.1M b-mercaptoethanol (Lane 2) or 0.5% Nonidet-P40 (lane 3) to the ATX sample has no effect on the size of the resultant protein band. Even complete denaturation of ATX of boiling the sample for 3 min in 0.1% SDS with (lane 5) or without (lane 4) β-mercaptoethanol, followed by addition of 0.5% Nonidet-P40 to maintain enzymatic activity, has no effect on the final size of deglycosylated protein, indicating that the deglycosidation reaction was complete even under mild conditions.

Because these results showed that ATX contained N-linked oligosaccharide groups, it became important to see if these sugar moieties were necessary for stimulation of motility. The partially purified ATX sample was treated with varying concentrations of PNGase F (0.1 to 60 mU/ml) under mild, non-denaturing conditions. Analysis of the resulting digest by polyacrylamide gel electrophoresis is shown in FIG. 17A. As this figure shows, the digestion was incomplete using from 0.1 to 10 mU/ml of enzyme and resulted in a smear of protein between 100-125 kDa. However, at higher concentrations of enzyme, cleavage of N-linked oligosaccharides from ATX appears to be complete. When these different digestion products were compared for their capacity to stimulate motility (FIG. 17B), there was no significant difference between groups.

Example 6 Cloning the 3' end of Autotaxin (4C11)

ATX is active in picomolar to nanomolar concentrations and is synthesized in very small concentrations by A2058 cells. As might be expected, the cDNA clone was relatively rare, requiring various strategies and multiple library screenings in order to identify it (FIG. 12). Attempts to utilize degenerate oligonucleotides deduced from known peptide sequences were unsuccessful--whether we used the oligo nucleotides for screening cDNA libraries or for reverse transcription of mRNA followed by amplification with the polymerase chain reaction (RT/PCR). We then utilized an affinity-purified anti-peptide ATX-102 antibodies to screen an A2058 expression library.

These anti-peptide antibodies were generated by methods well established in the art and described previously with slight modification (Wacher, et al., 1990). In brief, the previously identified peptide, ATX-102 (Stracke, et al., 1992), was synthesized on a Biosearch 9600 peptide synthesizer. It was then solubilized in 1×PBS containing 20% (v/v) DMSO and conjugated to the protein carrier, bovine serum albumin (BSA), with glutaraldehyde. For the first injection into New Zealand white rabbits, the BSA-peptide conjugate was emulsified with complete Freund's adjuvant and injected subcutaneously. For subsequent injections, the BSA-peptide conjugate was emulsified with incomplete Freund's adjuvant. The resultant antiserum was heat-inactivated at 56° C. for 30 min. Immunoglobulins were precipitated out in 47% saturated ammonium sulfate, then redissolved and dialyzed into PBS. Antibodies were adsorbed onto peptide-conjugated Affi-Gel 10 resin (made using the BioRad protocol), eluted with 0.1N acetic acid, and neutralized with 2M Tris-HCl, pH 8. The resulting affinity-purified antibodies were dialyzed into DPBS, concentrated, aliquotted, and stored at -20° C. The antibodies were found to recognize a 125 kDa protein on immunoblots of partially purified A2058 conditioned medium and to preferentially stain some breast carcinoma cells compared to normal breast using immunohistochemical techniques.

An A2058 cDNA library was prepared by purifying poly-A purified mRNA from the cells then size-selecting mRNA>1000 bp for the preparation of cDNA. The cDNA inserts were placed into λgt11 directionally, using the ProMega cDNA kit using standard methods well-established in the field. LE 392 cells were infected with the λgt11 and plaques were transferred onto nitrocellulose membranes by overnight incubation at 37° C. The antibody was incubated with the membranes in blocking buffer for 2 hr at room temperature, using approximately twice the concentration of antibody which gave a strong response on Western blot analysis. Secondary antibody was goat anti-rabbit immunoglobulin, and the blot was developed colorimetrically with 4-chloro-1-naphthol.

Positive clones were confirmed by antibody competition with specific peptides but not unrelated peptides. Using this technique and multiple subclonings, we obtained a partial cDNA clone of the autotaxin gene, which we called 4C11. The 4C11 insert was removed from λgt11 by restriction enzyme digests and subcloned into pBluescript for sequencing by standard Sanger techniques (Sanger, et al., 1977). The 4C11 clone contained bases, including the poly-adenylated tail and the AATAAA adenylation signal locus, i.e., it contained the 3' terminus of the gene. It also included a 627 base open reading frame. Database analysis of this nucleotide sequence revealed that it is unique. The predicated amino acid sequence for 4C11 is 209 amino acids long with exact matches for 7 previously identified ATX peptides: (ATX-20, ATX-34, ATX-102, ATX-104, ATX-204, ATX-215, and ATX-244).

Example 7 Cloning the 5' terminus of ATX

Database analysis of the 3' terminus of the ATX gene demonstrated a novel protein. However, we have found an interesting homology that has helped to guide us in exploring its function. ATX had a 45% amino acid identity and a 57% nucleotide identity with PC-1, a marker of B cell activation found on the surface of plasma cells. Using the PC-1 protein sequence as a guide, we found that ATX peptide homologies were scattered throughout the length of the protein. The only exception was the far amino terminus of PC-1, which includes the transmembrane and intracellular domains, and which had no homologies. Knowing approximate localization of the ATX peptides along the length of ATX, we then amplified different segments of ATX by the PCR (FIG. 13). These amplified segments of DNA were then subcloned into plasmids utilizing the TA Cloning kit of ProMega. The PCR amplified DNA could then be sequenced using standard Sanger sequencing techniques (Sanger, et al., 1977).

Cloning of full length ATX gene

A reverse transcriptase reaction was performed using total or oligo-(dT) purified RNA from A2058 or N-tera 2D1 cells as template and an anti-sense primer from the 5' end of 4C11 (GCTCAGATAAGGAGGAAAGAG). This was followed by one or two PCR amplification of the resultant cDNA using the commercially available kit from Perkin-Elmer and following manufacturer's directions. These PCR reactions utilized nested antisense primers from 4C11 (GAATCCGTAGGACATCTGCTT and TGTAGGCCAAACAGTTCTGAC) as well as degenerate, nested sense primers deduced from ATX peptides: ATX-101 (AAYTCIATGCARACIGTITTYGTIG and TTYGTIGGITAYGGICCIACITTYAA), ATX-103 (AAYTAYCTIACIAAYGTIGAYGAYAT and GAYGAYATIACICTIGTICCIGGIAC), or ATX-224 (TGYTTYGARYTICARGARGCIGGICCICC). The amplified DNA was then purified from a polyacrylamide gel using standard procedures and ligated into the pCR™ plasmid using the TA cloning kit (Invitrogen Corporation) according to manufacturer's directions.

The 5' RACE kit was utilized to extend the 5' end of ATX cDNA using total RNA from N-tera 2D1 as template and previously obtained sequence as primer (GCTGTCTTCAAACACAGC). The 5' end of the A2058 synthesized protein was obtained by using previously obtained sequence as primer (CTGGTGGCTGTAATCCATAGC) in a reverse transcriptase reaction with total A2058 RNA as template, followed by PCR amplification utilizing the 5' end of N-tera 2D1 sequence as sense primer (CGTGAAGGCAAAGAGAACACG) and a nested antisense primer (GCTGTCTTCAAACACAGC). A2058 DNA encoding ATX is set forth in a SEQ ID NO:68 and the amino acid sequence is provided in SEQ ID NO:69.

DNA sequencing:

DNA sequencing was performed using dideoxy methodology (Sanger, et al. 1977) and (³⁵ S)dATP (Du Pont, New England Nuclear).

We have found one region between the 5' end of the 4C11 and the ATX peptide designated ATX-101, also referred to as the "hot spot". This region has been sequenced five times with different sequences found each time. The hot spot appears to be located within the region from approximately nucleotide 1670 to 1815. The consensus sequence is represented by amino acids position 559 through 604. Variations found include DNA sequence that results in single and multiple amino acid insertions. One sequence had a stop codon in this region and may have represented an intron. This region has been found to be variable in forms of ATX.

Example 8 Cloning ATX in a human teratocarcinoma cell line

The fact that ATX is present in other cancer cells was confirmed by sequence information from N-tera 2D1, a human teratocarcinoma cell line. For these cells, a prepared cDNA library in λgt10 was amplified and the cDNA inserts were extracted. Using oligonucleotide primers based on known A2058 ATX sequence, DNA segments were amplified by PCR. The DNA segments were then subcloned into plasmids and sequenced as for A2058. We have 3104 bp DNA sequence for N-tera ATX (SEQ ID NO:66) and smaller portions thereof. This includes an open reading frame that codes for a putative protein containing 861 amino acids (SEQ ID NO:67) and smaller portions thereof. Like the A2058 ATX, the N-tera 2D1 sequence has homologies for multiple ATX peptides (FIG. 15). Sequence homology between the A2058 and N-tera 2D1 cells is approximately 99%.

Example 9 Cloning 5' end of ATX in human normal liver

The 5' end of ATX has proven difficult to obtain from either tumor cell line to date. Normal human liver mRNA was therefore amplified using the 5' RACE kit (Clontech) with known sequence from A2058 ATX as antisense primer. A DNA segment was obtained and has been sequenced. This segment codes for 979 amino acids, including an initiating methionine (SEQ ID NO:38). The putative protein sequence also includes a 20 amino acid transmembrane domain which is different from the tumor ATX's (SEQ ID NO:54), as shown in Table 7. Both tumorous forms of ATX apparently lack a transmembrane region and are instead secreted proteins.

                                      TABLE 7                                      __________________________________________________________________________     Nucleotide and Amino Acid Sequences Encoding Liver ATX Amino                   Terminus containing the Transmembrane region                                   __________________________________________________________________________     Protein Sequence (SEQ ID NO: 54)                                               Met Ala Arg Arg Ser Ser Phe Gln Ser Cys Gln Asp Ile Ser Leu Phe Thr Phe        Ala                                                                            Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala His Arg Ile Lys Arg Ala Glu        Gly                                                                            Trp                                                                            DNA Sequence (SEQ ID NO: 53)                                                   ATGGCAAGGA GGAGCTCGTT CCAGTCGTGT CAAGATATAT                                    CCCTGTTCAC                                                                     TTTTGCCGTT GGAGTCAATA TCTGCTTAGG ATTCACTGCA                                    CATCGAATTA                                                                     AGAGAGCAGA AGGATGG                                                             __________________________________________________________________________

Example 10 Domains of ATX

Searches of protein databases (Pearson, et. al. 1988) confirmed that the homology between ATX and PC-1 was present throughout the length of the extracellular portion of the molecules (Buckley, et. al., 1990; Funakoshi, et. al. 1992). There is a 45% amino acid identity and a 64% similarity between the 2 protein sequences (FIG. 18). For the cDNA sequence, the identity is ˜57%.

These proteins share several interesting properties and domains (FIG. 19). Both have a number of potential N-linked glycosylation sites: four for ATX (Asn54, Asn463, Asn577, Asn859) and nine for PC-1. Both have adjacent somatomedin B domains near the amino end of the extracellular domain. This somatomedin B domain is a cysteine-rich region containing 3 presumed cystine cross-linkages. ATX has 33 Cys residues and PC-1 has 37; 30 of these Cys residues are identical in placement. Both proteins also contain the loop region of an EF hand (Buckley, et. al. 1990; Kretsinger, 1987). In addition, both proteins have a transmembrane/signal peptide region with a short intracellular peptide, common in ectoenzymes (Maroux, 1987). However, the amino acid identity between ATX and PC-1 in the intracellular and transmembrane regions is only 11%.

Finally, both proteins have a region homologous to the bovine intestinal phosphodiesterase enzymatic domain with conversation of the threonine that is thought to act as the intermediate phosphate binding site (Culp, et al. 1985). PC-1 has been demonstrated to have phosphodiesterase type I, nucleotide pyrophosphatase, and threonine-specific kinase enzymatic activities (Rebbe, et al. 1991; Oda, et al. 1991). In order to test whether purified ATX had type I phosphodiesterase activity, samples were incubated with p-nitrophenyl thymidine-5'-monophosphate at pH 8.9 for 30 min. Samples were assayed in a 100 μl volume containing 50 mM Tris-HCl, pH 8.9 and 5 mM p-nitrophenyl thymidine-5'-monophosphate. After a 30 minute incubation at 37° C. the reactions were terminated by addition of 900 ml 0.1N NaOH and the amount of product formed was determined by reading the absorbance at 410 nm. ATX was found to hydrolyze the p-nitrophenyl thymidine-5'-monophosphate (Razzell, 1963) at a rate of 10 pmol/ng/min, a reaction rate similar to that reported for PC-1 (Oda, et al. 1993).

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All publications mentioned hereinabove are hereby incorporated in their entirety by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the present invention and appended claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 69                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5                                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        TrpHisValAlaArg                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 6                                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        ProLeuAspValTyrLys                                                             15                                                                             (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5                                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        TyrProAlaPheLys                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        GlnAlaGluValSer                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10                                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        ProGluGluValThrArgProAsnTyrLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9                                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        TyrAspValProTrpAsnGluThrIle                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10                                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        ValProProPheGluAsnIleGluLeuTyr                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11                                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        GlyGlyGlnProLeuTrpIleThrAlaThrLys                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16                                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        ValAsnSerMetGlnThrValPheValGlyTyrGly                                           1510                                                                           ProThrPheLys                                                                   15                                                                             (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12                                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       AspIleGluHisLeuThrSerLeuAspPhePheArg                                           1510                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23                                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       ThrGluPheLeuSerAsnTyrLeuThrAsnValAsp                                           1510                                                                           AspIleThrLeuValProGluThrLeuGlyArg                                              1520                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       GTTGGCAGCNACRTGCCA18                                                           (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       TGGCAYGTNGCTGCCAAC18                                                           (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       CTTGAAGGCAGGGTA15                                                              (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       TAYCCTGCNTTYAAG15                                                              (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       GGTNACYTCYTCAGG15                                                              (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       CCTGARGARGTNACC15                                                              (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       NGTNGCRTCRAATGGCACRTC21                                                        (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       GAYGTGCCATTYGAYGCNACN21                                                        (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       GTTDATRTTSTCRAATGGGGG21                                                        (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       CCCCCATTTGAGAACATCAAC21                                                        (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       CTTNGTNGCNGTDATCCANARGGGYTGGCCGCC33                                            (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       GGCGGCCARCCCYTNTGGATHACNGCNACNAAG33                                            (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       CTTRAAGGTGGGGCCRTAGCCCACRAAGACTGTYTGCAT39                                      (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       ATGCARACAGTCTTYGTGGGCTAYGGCCCCACCTTYAAR39                                      (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9                                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       GlnTyrLeuHisGlnTyrGlySerSer                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5                                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       ValLeuAsnTyrPhe                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5                                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       TyrLeuAsnAlaThr                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       HisLeuLeuTyrGlyArgProAlaValLeuTyr                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       SerTyrProGluIleLeuThrProAlaAspAsn                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       XaaTyrGlyPheLeuPheProProTyrLeuSerSer                                           1510                                                                           SerPro                                                                         (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       ThrPheProAsnLeuTyrThrPheAlaThrGlyLeu                                           1510                                                                           Tyr                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       ValAsnValIleSerGlyProIleAspAspTyrAsp                                           1510                                                                           TyrAspGlyLeuHisAspThrGluAspLys                                                 1520                                                                           (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 829                                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: No                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE:                                                               (G) CELL TYPE: Melanoma                                                        (H) CELL LINE: A2058                                                           (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Putative protein                                        sequence of A2058 Autotaxin                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       CysHisAspPheAspGluLeuCysLeuLysThrAla                                           1510                                                                           ArgGlyTrpGluCysThrLysAspArgCysGlyGlu                                           1520                                                                           ValArgAsnGluGluAsnAlaCysHisCysSerGlu                                           253035                                                                         AspCysLeuAlaArgGlyAspCysCysThrAsnTyr                                           4045                                                                           GlnValValCysLysGlyGluSerHisTrpValAsp                                           505560                                                                         AspAspCysGluGluIleLysAlaAlaGluCysPro                                           6570                                                                           AlaGlyPheValArgProProLeuIleIlePheSer                                           7580                                                                           ValAspGlyPheArgAlaSerTyrMetLysLysGly                                           859095                                                                         SerLysValMetProAsnIleGluLysLeuArgSer                                           100105                                                                         CysGlyThrHisSerProTyrMetArgProValTyr                                           110115120                                                                      ProThrLysThrPheProAsnLeuTyrThrLeuAla                                           125130                                                                         ThrGlyLeuTyrProGluSerHisGlyIleValGly                                           135140                                                                         AsnSerMetTyrAspProValPheAspAlaThrPhe                                           145150155                                                                      HisLeuArgGlyArgGluLysPheAsnHisArgTrp                                           160165                                                                         TrpGlyGlyGlnProLeuTrpIleThrAlaThrLys                                           170175180                                                                      GlnGlyValLysAlaGlyThrPhePheTrpSerVal                                           185190                                                                         ValIleProHisGluArgArgIleLeuThrIleLeu                                           195200                                                                         ArgTrpLeuThrLeuProAspHisGluArgProSer                                           205210215                                                                      ValTyrAlaPheTyrSerGluGlnProAspPheSer                                           220225                                                                         GlyHisLysTyrGlyProPheGlyProGluGluSer                                           230235240                                                                      SerTyrGlySerProPheThrProAlaLysArgPro                                           245250                                                                         LysArgLysValAlaProLysArgArgGlnGluArg                                           255260                                                                         ProValAlaProProLysLysArgArgArgLysIle                                           265270275                                                                      HisArgMetAspHisTyrAlaAlaGluThrArgGln                                           280285                                                                         AspLysMetThrAsnProLeuArgGluIleAspLys                                           290295300                                                                      IleValGlyGlnLeuMetAspGlyLeuLysGlnLeu                                           305310                                                                         LysLeuArgArgCysValAsnValIlePheValGly                                           315320                                                                         AspHisGlyMetGluAspValThrCysAspArgThr                                           325330335                                                                      GluPheLeuSerAsnTyrLeuThrAsnValAspAsp                                           340345                                                                         IleThrLeuValProGlyThrLeuGlyArgIleArg                                           350355360                                                                      SerLysPheSerAsnAsnAlaLysTyrAspProLys                                           365370                                                                         AlaIleIleAlaAsnLeuThrCysLysLysProAsp                                           375380                                                                         GlnHisPheLysProTyrLeuLysGlnHisLeuPro                                           385390395                                                                      LysArgLeuHisTyrAlaAsnAsnArgArgIleGlu                                           400405                                                                         AspIleHisLeuLeuValGluArgArgTrpHisVal                                           410415420                                                                      AlaArgLysProLeuAspValTyrLysLysProSer                                           425430                                                                         GlyLysCysPhePheGlnGlyAspHisGlyPheAsp                                           435440                                                                         AsnLysValAsnSerMetGlnThrValPheValGly                                           445450455                                                                      TyrGlyProThrPheLysTyrLysThrLysValPro                                           460465                                                                         ProPheGluAsnIleGluLeuTyrAsnValMetCys                                           470475480                                                                      AspLeuLeuGlyLeuLysProAlaProAsnAsnGly                                           485490                                                                         ThrHisGlySerLeuAsnHisLeuLeuArgThrAsn                                           495500                                                                         ThrPheArgProThrMetProGluGluValThrArg                                           505510515                                                                      ProAsnTyrProGlyIleMetTyrLeuGlnSerAsp                                           520525                                                                         AspAspLeuGlyCysThrCysAspAspLysValGlu                                           530535540                                                                      ProLysAsnLysLeuAspGluLeuAsnLysArgLeu                                           545550                                                                         HisThrLysGlySerThrGluGluArgHisLeuLeu                                           555560                                                                         TyrGlyArgProAlaValLeuTyrArgThrArgTyr                                           565570575                                                                      AspIleLeuTyrHisThrAspPheGluSerGlyTyr                                           580585                                                                         SerGluIlePheLeuMetLeuLeuTrpThrSerTyr                                           590595600                                                                      ThrValSerLysGlnAlaGluValSerSerValPro                                           605610                                                                         AspHisLeuThrSerCysValArgProAspValArg                                           615620                                                                         ValSerProSerPheSerGlnAsnCysLeuAlaTyr                                           625630635                                                                      LysAsnAspLysGlnMetSerTyrGlyPheLeuPhe                                           640645                                                                         ProProTyrLeuSerSerSerProGluAlaLysTyr                                           650655660                                                                      AspAlaPheLeuValThrAsnMetValProMetTyr                                           665670                                                                         ProAlaPheLysArgValTrpAsnTyrPheGlnArg                                           675680                                                                         ValLeuValLysLysTyrAlaSerGluArgAsnGly                                           685690695                                                                      ValAsnValIleSerGlyProIlePheAspTyrAsp                                           700705                                                                         TyrAspGlyLeuHisAspThrGluAspLysIleLys                                           710715720                                                                      GlnTyrValGluGlySerSerIleProValProThr                                           725730                                                                         HisTyrTyrSerIleIleThrSerCysLeuAspPhe                                           735740                                                                         ThrGlnProAlaAspLysCysAspGlyProLeuSer                                           745750755                                                                      ValSerSerPheIleLeuProHisArgProAspAsn                                           760765                                                                         GluGluSerCysAsnSerSerGluAspGluSerLys                                           770775780                                                                      TrpValGluGluLeuMetLysMetHisThrAlaArg                                           785790                                                                         ValArgAspIleGluHisLeuThrSerLeuAspPhe                                           795800                                                                         PheArgLysThrSerArgSerTyrProGluIleLeu                                           805810815                                                                      ThrLeuLysThrTyrLeuHisThrTyrGluSerGlu                                           820825                                                                         Ile                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2946                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE:                                                               (G) CELL TYPE: Melanoma                                                        (H) CELL LINE: A2058                                                           (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Partial DNA Sequence                                    of A2058 Autotaxin                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       GCTGCCATGACTTTGATGAGCTGTGTTTGAAGACAGCCCG40                                     TGGCTGGGAGTGTACTAAGGACAGATGTGGAGAAGTCAGA80                                     AATGAAGAAAATGCCTGTCACTGCTCAGAGGACTGCTTGG120                                    CCAGGGGAGACTGCTGTACCAATTACCAAGTGGTTTGCAA160                                    AGGAGAGTCGCATTGGGTTGATGATGACTGTGAGGAAATA200                                    AAGGCCGCAGAATGCCCTGCAGGGTTTGTTCGCCCTCCAT240                                    TAATCATCTTCTCCGTGGATGGCTTCCGTGCATCATACAT280                                    GAAGAAAGGCAGCAAAGTCATGCCTAATATTGAAAAACTA320                                    AGGTCTTGTGGCACACACTCTCCCTACATGAGGCCGGTGT360                                    ACCCAACTAAAACCTTTCCTAACTTATACACTTTGGCCAC400                                    TGGGCTATATCCAGAATCACATGGAATTGTTGGCAATTCA440                                    ATGTATGATCCTGTATTTGATGCCACTTTTCATCTGCGAG480                                    GGCGAGAGAAATTTAATCATAGATGGTGGGGAGGTCAACC520                                    GCTATGGATTACAGCCACCAAGCAAGGGGTGAAAGCTGGA560                                    ACATTCTTTTGGTCTGTTGTCATCCCTCACGAGCGGAGAA600                                    TATTAACCATATTGCGGTGGCTCACCCTGCCAGATCATGA640                                    GAGGCCTTCGGTCTATGCCTTCTATTCTGAGCAACCTGAT680                                    TTCTCTGGACACAAATATGGCCCTTTCGGCCCTGAGGAGA720                                    GTAGTTATGGCTCACCTTTTACTCCGGCTAAGAGACCTAA760                                    GAGGAAAGTTGCCCCTAAGAGGAGACAGGAAAGACCAGTT800                                    GCTCCTCCAAAGAAAAGAAGAAGAAAAATACATAGGATGG840                                    ATCATTATGCTGCGGAAACTCGTCAGGACAAAATGACAAA880                                    TCCTCTGAGGGAAATCGACAAAATTGTGGGGCAATTAATG920                                    GATGGACTGAAACAACTAAAACTGCGTCGGTGTGTCAACG960                                    TCATCTTTGTCGGAGACCATGGAATGGAAGATGTCACATG1000                                   TGATAGAACTGAGTTCTTGAGTAATTACCTAACTAATGTG1040                                   GATGATATTACTTTAGTGCCTGGAACTCTAGGAAGAATTC1080                                   GATCCAAATTTAGCAACAATGCTAAATATGACCCCAAAGC1120                                   CATTATTGCCAATCTCACGTGTAAAAAACCAGATCAGCAC1160                                   TTTAAGCCTTACTTGAAACAGCACCTTCCCAAACGTTTGC1200                                   ACTATGCCAACAACAGAAGAATTGAGGATATCCATTTATT1240                                   GGTGGAACGCAGATGGCATGTTGCAAGGAAACCTTTGGAT1280                                   GTTTATAAGAAACCATCAGGAAAATGCTTTTTCCAGGGAG1320                                   ACCACGGATTTGATAACAAGGTCAACAGCATGCAGACTGT1360                                   TTTTGTAGGTTATGGCCCAACATTTAAGTACAAGACTAAA1400                                   GTGCCTCCATTTGAAAACATTGAACTTTACAATGTTATGT1440                                   GTGATCTCCTGGGATTGAAGCCAGCTCCTAATAATGGGAC1480                                   CCATGGAAGTTTGAATCATCTCCTGCGCACTAATACCTTC1520                                   AGGCCAACCATGCCAGAGGAAGTTACCAGACCCAATTATC1560                                   CAGGGATTATGTACCTTCAGTCTGATTTTGACCTGGGCTG1600                                   CACTTGTGATGATAAGGTAGAGCCAAAGAACAAGTTGGAT1640                                   GAACTCAACAAACGGCTTCATACAAAAGGGTCTACAGAAG1680                                   AGAGACACCTCCTCTATGGGCGACCTGCAGTGCTTTATCG1720                                   GACTAGATATGATATCTTATATCACACTGACTTTGAAAGT1760                                   GGTTATAGTGAAATATTCCTAATGCTACTCTGGACATCAT1800                                   ATACTGTTTCCAAACAGGCTGAGGTTTCCAGCGTTCCTGA1840                                   CCATCTGACCAGTTGCGTCCGGCCTGATGTCCGTGTTTCT1880                                   CCGAGTTTCAGTCAGAACTGTTTGGCCTACAAAAATGATA1920                                   AGCAGATGTCCTACGGATTCCTCTTTCCTCCTTATCTGAG1960                                   CTCTTCACCAGAGGCTAAATATGATGCATTCCTTGTAACC2000                                   AATATGGTTCCAATGTATCCTGCTTTCAAACGGGTCTGGA2040                                   ATTATTTCCAAAGGGTATTGGTGAAGAAATATGCTTCGGA2080                                   AAGAAATGGAGTTAACGTGATAAGTGGACCAATCTTCGAC2120                                   TATGACTATGATGGCTTACATGACACAGAAGACAAAATAA2160                                   AACAGTACGTGGAAGGCAGTTCCATTCCTGTTCCAACTCA2200                                   CTACTACAGCATCATCACCAGCTGTCTGGATTTCACTCAG2240                                   CCTGCCGACAAGTGTGACGGCCCTCTCTCTGTGTCCTCCT2280                                   TCATCCTGCCTCACCGGCCTGACAAAGAGGAGAGCTGCAA2320                                   TAGCTCAGAGGACGAATCAAAATGGGTAGAAGAACTCATG2360                                   AAGATGCACACAGCTAGGGTGCGTGACATTGAACATCTCA2400                                   CCAGCCTGGACTTCTTCCGAAAGACCAGCCGCAGCTACCC2440                                   AGAAATCCTGACACTCAAGACATACCTGCATACATATGAG2480                                   AGCGAGATTTAACTTTCTGAGCATCTGCAGTACAGTCTTA2520                                   TCAACTGGTTGTATATTTTTATATTGTTTTTGTATTTATT2560                                   AATTTGAAACCAGGACATTAAAAATGTTAGTATTTTAATC2600                                   CTGTACCAAATCTGACATATTATGCCTGAATGACTCCACT2640                                   GTTTTTCTCTAATGCTTGATTTAGGTAGCCTTGTGTTCTG2680                                   AGTAGAGCTTGTAATAAATACTGCAGCTTGAGAAAAAGTG2720                                   GAAGCTTCTAAATGGTGCTGCAGATTTGATATTTGCATTG2760                                   AGGAAATATTAATTTTCCAATGCACAGTTGCCACATTTAG2800                                   TCCTGTACTGTATGGAAACACTGATTTTGTAAAGTTGCCT2840                                   TTATTTGCTGTTAACTGTTAACTATGACAGATATATTTAA2880                                   GCCTTATAAACCAATCTTAAACATAATAAATCACACATTC2920                                   AGTTTTAAAAAAAAAAAAAAAAAAAA2946                                                 (2) INFORMATION FOR SEQ ID NO:36:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 788                                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: No                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE:                                                               (G) CELL TYPE: teratocarcinoma                                                 (H) CELL LINE: N-tera 2D1                                                      (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: N-tera 2D1 putative                                     ATX protein sequence                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       CysAspAsnLeuCysLysSerTyrThrSerCysCys                                           1510                                                                           HisAspPheAspGluLeuCysLeuLysThrAlaArg                                           1520                                                                           AlaTrpGluCysThrLysAspArgCysGlyGluVal                                           253035                                                                         ArgAsnGluGluAsnAlaCysHisCysSerGluAsp                                           4045                                                                           CysLeuAlaArgGlyAspCysCysThrAsnTyrGln                                           505560                                                                         ValValCysLysGlyGluSerHisTrpValAspAsp                                           6570                                                                           AspCysGluGluIleLysAlaAlaGluCysLeuGln                                           7580                                                                           ValAspSerProSerIleAsnHisLeuLeuArgGly                                           859095                                                                         TrpLeuProMetThrSerTyrMetLysLysGlySer                                           100105                                                                         LysValMetProAsnIleGluLysLeuArgSerCys                                           110115120                                                                      GlyThrHisSerProTyrMetArgProValTyrPro                                           125130                                                                         ThrLysThrPheProAsnLeuTyrThrLeuAlaThr                                           135140                                                                         GlyLeuTyrProGluSerHisGlyIleValGlyAsn                                           145150155                                                                      SerMetTyrAspProValPheAspAlaThrPheHis                                           160165                                                                         LeuArgGlyArgGluLysPheAsnHisArgTrpTrp                                           170175180                                                                      AlaGlyGlnProLeuTrpIleThrAlaThrLysGln                                           185190                                                                         ArgGlyGluSerTrpAsnIleLeuLeuValCysCys                                           195200                                                                         HisProSerArgAlaGluIleLeuThrIleLeuGln                                           205210215                                                                      TrpLeuThrLeuProAspHisGluArgProSerVal                                           220225                                                                         TyrAlaPheTyrSerGluGlnProAspPheSerGly                                           230235240                                                                      HisLysHisMetProPheGlyProGluMetProAsn                                           245250                                                                         ProLeuArgGluMetHisLysIleValGlyGlnLeu                                           255260                                                                         MetAspGlyLeuLysGlnLeuLysLeuHisArgCys                                           265270275                                                                      ValAsnValIlePheValGluThrMetAspGlyArg                                           280285                                                                         CysHisMetTyrArgThrGluPheLeuSerAsnTyr                                           290295300                                                                      LeuThrAsnValAspAspIleThrLeuValProGly                                           305310                                                                         ThrLeuGlyArgIleArgSerLysPheSerAsnAsn                                           315320                                                                         AlaLysTyrAspProLysAlaIleIleAlaAsnLeu                                           325330335                                                                      ThrCysLysLysProAspGlnHisPheLysProTyr                                           340345                                                                         LeuLysGlnHisLeuProLysArgLeuHisTyrAla                                           350355360                                                                      AsnAsnArgArgIleGluAspIleHisLeuLeuVal                                           365370                                                                         GluArgArgTrpHisValAlaArgLysProLeuAsp                                           375380                                                                         ValTyrLysLysProSerGlyAsnAlaPheSerArg                                           385390395                                                                      GluThrThrAlaPheAspAsnLysValAsnSerMet                                           400405                                                                         GlnThrValPheValGlyTyrGlyProThrPheLys                                           410415420                                                                      TyrLysThrLysValProProPheGluAsnIleGlu                                           425430                                                                         LeuTyrAsnValMetCysAspLeuLeuGlyLeuLys                                           435440                                                                         ProAlaProAsnAsnGlyThrHisPheSerLeuAsn                                           445450455                                                                      HisLeuLeuArgThrAsnThrPheArgProThrMet                                           460465                                                                         ProGluGluValThrArgProAsnTyrProGlyIle                                           470475480                                                                      MetTyrLeuGlnSerAspPheAspLeuGlyCysThr                                           485490                                                                         CysAspAspLysValGluProLysAsnLysLeuAsp                                           495500                                                                         GluLeuAsnLysArgLeuHisThrLysGlySerThr                                           505510515                                                                      GluGluArgHisLeuLeuTyrGlyAspArgProAla                                           520525                                                                         ValLeuTyrArgThrArgTyrAspIleLeuTyrHis                                           530535540                                                                      ThrAspPheGluSerGlyTyrSerGluIlePheLeu                                           545550                                                                         MetProLeuTrpThrSerTyrThrValSerLysGln                                           555560                                                                         AlaGluValSerSerValProAspHisLeuThrSer                                           565570575                                                                      CysValArgProAspValArgValSerProSerPhe                                           580585                                                                         SerGlnAsnCysLeuAlaTyrLysAsnAspLysGln                                           590595600                                                                      MetSerTyrGlyGlyLeuGlyProProTyrLeuSer                                           605610                                                                         SerSerProGluAlaLysTyrAspAlaPheLeuVal                                           615620                                                                         ThrAsnMetValProMetTyrProAlaPheLysArg                                           625630635                                                                      ValTrpAsnTyrPheGlnArgValLeuValLysLys                                           640645                                                                         TyrAlaSerGluArgAsnGlyValAsnValIleSer                                           650655660                                                                      GlyProIlePheAspTyrAspTyrAspGlyLeuHis                                           665670                                                                         AspThrGluAspLysIleLysGlnTyrValGluGly                                           675680                                                                         SerSerIleProValProThrHisTyrTyrSerIle                                           685690695                                                                      IleThrSerCysLeuAspPheThrGlnProAlaAsp                                           700705                                                                         LysCysAspGlyProLeuSerValSerSerPheIle                                           710715720                                                                      LeuProHisArgProAspAsnGluGluSerCysAsn                                           725730                                                                         SerSerGluAspGluSerLysTrpValGluGluLeu                                           735740                                                                         MetLysMetHisThrAlaArgValArgAspIleGlu                                           745750755                                                                      HisLeuThrSerLeuAspPhePheArgLysThrSer                                           760765                                                                         ArgSerTyrProGluIleLeuThrLeuLysThrTyr                                           770775780                                                                      LeuHisThrTyrGluSerGluIle                                                       785                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2712                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE:                                                               (G) CELL TYPE: teratocarcinoma                                                 (H) CELL LINE: N-tera 2D1                                                      (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: N-tera 2D1 ATX DNA                                      sequence                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                       TGTGACAACTTGTGTAAGAGCTATACCAGTTGCTGCCATG40                                     ACTTTGATGAGCTGTGTTTGAAGACAGCCCGTGCGTGGGA80                                     GTGTACTAAGGACAGATGTGGGGAAGTCAGAAATGAAGAA120                                    AATGCCTGTCACTGCTCAGAGGACTGCTTGGCCAGGGGAG160                                    ACTGCTGTAACAATTACCAAGTGGTTTGCAAAGGAGAGTC200                                    GCATTGGGTTGATGATGACTGTGAGGAAATAAAGGCCGCA240                                    GAATGCCTGCAGGTTTGTTCGCCCTCCATTAATCATCTTC280                                    TCCGTGGATGGCTTCCGATGACATCATACATGAAGAAAGG320                                    CAGCAAAGTCATGCCTAATATTGAAAAACTAAGGTCTTGT360                                    GGCACACACTCTCCCTACATGAGGCCGGTGTACCCAACTA400                                    AAACCTTTCCTAACTTATACACTTTGGCCACTGGGCTATA440                                    TCCAGAATCACATGGAATTGTTGGCAATTCAATGTATGAT480                                    CCTGTATTTGATGCCACTTTTCATCTGCGAGGGCGAGAGA520                                    AATTTAATCATAGATGGTGGGGAGGTCAACCGCTATGGAT560                                    TACAGCCACCAAGCAAAGGGGTGAAAGCTGGAACATTCTT600                                    TTGGTCTGTTGTCATCCCTCACGAGCGGAGATATTAACCA640                                    TATTGCAGTGGCTCACCCTGCCAGATCATGAGAGGCCTTC680                                    GGTCTATGCCTTCTATTCTGAGCAACCTGATTTCTCTGGA720                                    CACAAACATATGCCTTTCGGCCCTGAGATGACAAATCCTC760                                    TGAGGGAAATGCACAAAATTGTGGGGCAATTAATGGATGG800                                    ACTGAAACAACTAAAACTGCATCGGTGTGTCAACGTCATC840                                    TTTGTCGAGACCATGGATGGAAGATGTCACATGTATAGAA880                                    CTGAGTTCTTGAGTAATTACCTAACTAATGTGGATGATAT920                                    TACTTTAGTGCCTGGAACTCTAGGAAGAATTCGATCCAAA960                                    TTTAGCAACAATGCTAAATATCACCCCAAAGCCATTATTG1000                                   CCAATCTCACGTGTAAAAAACCAGATCAGCACTTTAAGCC1040                                   TTACTTGAAACAGCACCTTCCCAAACGTTTGCACTATGCC1080                                   AACAACAGAAGAATTGAGGATATCCATTTATTGGTGGAAC1120                                   GCAGATGGCATGTTGCAAGGAAACCTTTGGATGTTTATAA1160                                   GAAACCATCAGGAAATGCTTTTTCCAGGGAGACCACGGCA1200                                   TTTGATAACAAGGTCAACAGCATGCAGACTGTTTTTGTAG1240                                   GTTATGGCCCAACATTTAAGTACAAGACTAAAGTDCCTCC1280                                   ATTTGAAAACATTGAACTTTAAAATGTTATGTGTGATCTC1320                                   CTGGGATTGAAGCCAGCTCCTAATAATGGGACCCATGGAA1360                                   GTTTGAATCATCTCCTGCGCACTAATACCTTCAGGCCAAC1400                                   CATGCCAGAGGAAGTTACCAGACCCTATTATCCAGGGATT1440                                   ATGTACCTTCAGTCTGATTTTGACCTGGGCTGCACTTGTG1480                                   ATGATAAGGTAGAGCCAAAGAACAAGTTGGATGAACTCAA1520                                   CAAACGGCTTCATACAAAAGGGTCTACAGAAGAGAGACAC1560                                   CTCCTCTATGGGGATCGACCTGCAGTGCTTTATCGGACTA1600                                   GATATGATATCTTATATCACACTGACTTTGAAAGTGGTTA1640                                   TAGTGAAATATTCCTAATGCCACTCTGGACATCATATACT1680                                   GTTTCCAAACAGGCTGAGGTTTCCAGCGTTCCTGACCATC1720                                   TGACCAGTTGCGTCCGGCCTGATGTCCGTGTTTCTCCGAG1760                                   TTTCAGTCAGAACTGTTTGGCCTACAAAAATGATAAGCAG1800                                   ATGTCCTACGGATTCCTCTTTCCTCCTTATCTGAGCTCTT1840                                   CACCAGAGGCTAAATATGATGCATTCCTTGTAACCAATAT1880                                   GGTTCCAATGTATCCTGCTTTCAAACGGGTCTGGAATTAT1920                                   TTCCAAAGGGTATTGGTGAAGAAATATGCTTCGGAAAGAA1960                                   ATGGAGTTAACGTGATAAGTGGACCAATCTTCGACTATGA2000                                   CTATGATGGCTTACATGACACAGAAGACAAAATAAAACAG2040                                   TACGTGGAAGGCAGTTCCATTCCTGTTCCAACTCACTACT2080                                   ACAGCATCATCACCAGCTGTCTGGATTTCACTCAGCCTGC2120                                   CGACAAGTGTGACGGCCCTCTCTCTGTGTCCTCCTTCATC2160                                   CTGCCTCACCGGCCTGACAACGAGGAGAGCTGCAATAGCT2200                                   CAGAGGACGAATCAAAATGGGTAGAAGAACTCATGAAGAT2240                                   GCACACAGCTAGGGTGCGTGACATTGAACATCTCACCAGC2280                                   CTGGACTTCTTCCGAAAGACCAGCCGCAGCTACCCAGAAA2320                                   TCCTGACACTCAAGACATACCTGCATACATATGAGAGCGA2360                                   GATTTAACTTTCTGAGCATCTGCAGTACAGTCTTATCAAC2400                                   TGGTTGTATATTTTTATATTGTTTTTGTATTTATTAATTT2440                                   GAAACCAGGACATTAAAAATGTTAGTATTTTAATCCTGTA2480                                   CCAAATCTGACATATTATGCCTGAATGACTCCACTGTTTT2520                                   TCTCTAATGCTTGATTTAGGTAGCCTTGTGTTCTGAGTAG2560                                   AGCTTGTAATAAATACTGCAGCTTGAGTTTTTAGTGGAAG2600                                   CTTCTAAATGGTGCTGCAGATTTGATATTTGCATTGAGGA2640                                   AATATTAATTTTCCAATGCACAGTTGCCACATTTAGTCCT2680                                   GTACTGTATGGAAACACTGATTTTGTAAAGTT2712                                           (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 979                                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: No                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE: Liver                                                         (G) CELL TYPE:                                                                 (H) CELL LINE:                                                                 (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: putative autotaxin                                      protein sequence from human liver                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                       MetAlaArgArgSerSerPheGlnSerCysGlnAsp                                           1510                                                                           IleSerLeuPheThrPheAlaValGlyValAsnIle                                           1520                                                                           CysLeuGlyPheThrAlaHisArgIleLysArgAla                                           253035                                                                         GluGlyTrpGluGluGlyProProThrValLeuSer                                           4045                                                                           AspSerProTrpThrAsnIleSerGlySerCysLys                                           505560                                                                         GlyArgCysPheGluLeuGlnGluAlaGlyProPro                                           6570                                                                           AspCysArgCysAspAsnLeuCysLysSerTyrThr                                           7580                                                                           SerCysCysHisAspPheAspGluLeuCysLeuLys                                           859095                                                                         ThrAlaArgAlaTrpGluCysThrLysAspArgCys                                           100105                                                                         GlyGluValArgAsnGluGluAsnAlaCysHisCys                                           110115120                                                                      SerGluAspCysLeuAlaArgGlyAspCysCysThr                                           125130                                                                         AsnTyrGlnValValCysLysGlyGluSerHisTrp                                           135140                                                                         ValAspAspAspCysGluGluIleLysAlaAlaGlu                                           145150155                                                                      CysLeuGlnValCysSerProSerIleAsnHisLeu                                           160165                                                                         LeuArgGlyTrpLeuProMetThrSerTyrMetLys                                           170175180                                                                      LysGlySerLysValMetProAsnIleGluLysLeu                                           185190                                                                         ArgSerCysGlyThrHisSerProTyrMetArgPro                                           195200                                                                         ValTyrProThrLysThrPheProAsnLeuTyrThr                                           205210215                                                                      LeuAlaThrGlyLeuTyrProGluSerHisGlyIle                                           220225                                                                         ValGlyAsnSerMetTyrAspProValPheAspAla                                           230235240                                                                      ThrPheHisLeuArgGlyArgGluLysPheAsnHis                                           245250                                                                         ArgTrpTrpGlyGlyGlnProLeuTrpIleThrAla                                           255260                                                                         ThrLysGlnArgGlyGluSerTrpAsnIleLeuLeu                                           265270275                                                                      ValCysCysHisProSerArgAlaGluIleLeuThr                                           280285                                                                         IleLeuGlnTrpLeuThrLeuProAspHisGluArg                                           290295300                                                                      ProSerValTyrAlaPheTyrSerGluGlnProAsp                                           305310                                                                         PheSerGlyHisLysHisMetProPheGlyProGlu                                           315320                                                                         MetThrAsnProLeuArgGluMetHisLysIleVal                                           325330335                                                                      GlyGlnLeuMetAspGlyLeuLysGlnLeuLysLeu                                           340345                                                                         HisArgCysValAsnValIlePheValGluThrMet                                           350355360                                                                      AspGlyArgCysHisMetTyrArgThrGluPheLeu                                           365370                                                                         SerAsnTyrLeuThrAsnValAspAspIleThrLeu                                           375380                                                                         ValProGlyThrLeuGlyArgIleArgSerLysPhe                                           385390395                                                                      SerAsnAsnAlaLysTyrAspProLysAlaIleIle                                           400405                                                                         AlaAsnLeuThrCysLysLysProAspGlnHisPhe                                           410415420                                                                      LysProTyrLeuLysGlnHisLeuProLysArgLeu                                           425430                                                                         HisTyrAlaAsnAsnArgArgIleGluAspIleHis                                           435440                                                                         LeuLeuValGluArgArgTrpHisValAlaArgLys                                           445450455                                                                      ProLeuAspValTyrLysLysProSerGlyAsnAla                                           460465                                                                         PheSerArgGluThrThrAlaPheAspAsnLysVal                                           470475480                                                                      AsnSerMetGlnThrValPheValGlyTyrGlyPro                                           485490                                                                         ThrPheLysTyrLysThrLysValProProPheGlu                                           495500                                                                         AsnIleGluLeuTyrAsnValMetCysAspLeuLeu                                           505510515                                                                      GlyLeuLysProAlaProAsnAsnGlyThrHisGly                                           520525                                                                         SerLeuAsnHisLeuLeuArgThrAsnThrPheArg                                           530535540                                                                      ProThrMetProGluGluValThrArgProAsnTyr                                           545550                                                                         ProGlyIleMetTyrLeuGlnSerAspPheAspLeu                                           555560                                                                         GlyCysThrCysAspAspLysValGluProLysAsn                                           565570575                                                                      LysLeuAspGluLeuAsnLysArgLeuHisThrLys                                           580585                                                                         GlySerThrGluGluArgHisLeuLeuTyrGlyAsp                                           590595600                                                                      ArgProAlaValLeuTyrArgThrArgTyrAspIle                                           605610                                                                         LeuTyrHisThrAspPheGluSerGlyTyrSerGlu                                           615620                                                                         IlePheLeuMetProLeuTrpThrSerTyrThrVal                                           625630635                                                                      SerLysGlnAlaGluValSerSerValProAspHis                                           640645                                                                         LeuThrSerCysValArgProAspValArgValSer                                           650655660                                                                      ProSerPheSerGlnAsnCysLeuAlaTyrLysAsn                                           665670                                                                         AspLysGlnMetSerTyrGlyPheLeuPheProPro                                           675680                                                                         TyrLeuSerSerSerProGluAlaLysTyrAspAla                                           685690695                                                                      PheLeuValThrAsnMetValProMetTyrProAla                                           700705                                                                         PheLysArgValTrpAsnTyrPheGlnArgValLeu                                           710715720                                                                      ValLysLysTyrAlaSerGluArgAsnGlyValAsn                                           725730                                                                         ValIleSerGlyProIlePheAspTyrAspTyrAsp                                           735740                                                                         GlyLeuHisAspThrGluAspLysIleLysGlnTyr                                           745750755                                                                      ValGluGlySerSerIleProValProThrHisTyr                                           760765                                                                         TyrSerIleIleThrSerCysLeuAspPheThrGln                                           770775780                                                                      ProAlaAspLysCysAspGlyProLeuSerValSer                                           785790                                                                         SerPheIleLeuProHisArgProAspAsnGluGlu                                           795800                                                                         SerCysAsnSerSerGluAspGluSerLysTrpVal                                           805810815                                                                      GluGluLeuMetLysMetHisThrAlaArgValArg                                           820825                                                                         AspIleGluHisLeuThrSerLeuAspPhePheArg                                           830835840                                                                      LysThrSerArgSerTyrProGluIleLeuThrLeu                                           845850                                                                         LysThrTyrLeuHisThrTyrGluSerGluIleXaa                                           855860                                                                         LeuSerGluHisLeuGlnTyrSerLeuIleAsnTrp                                           865870875                                                                      LeuTyrIlePheIleLeuPheLeuTyrLeuLeuIle                                           880885                                                                         XaaAsnGlnAspIleLysAsnValSerIleLeuIle                                           890895900                                                                      LeuTyrGlnIleXaaHisIleMetProGluXaaLeu                                           905910                                                                         HisCysPheSerLeuMetLeuAspLeuGlySerLeu                                           915920                                                                         ValPheXaaValGluLeuValIleAsnThrAlaAla                                           925930935                                                                      XaaValPheSerGlySerPheXaaMetValLeuGln                                           940945                                                                         IleXaaTyrLeuHisXaaGlyAsnIleAsnPhePro                                           950955960                                                                      MetHisSerCysHisIleXaaSerCysThrValTrp                                           965970                                                                         LysHisXaaPheCysLysVal                                                          975                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:8                                                                   (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-204                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       MetHisThrAlaArgValArgAsp                                                       (2) INFORMATION FOR SEQ ID NO:40:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8                                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-205                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                       PheSerAsnAsnAlaLysTyrAsp                                                       5                                                                              (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7                                                                  (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-209                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                       ValMetProAsnIleGluLys                                                          5                                                                              (2) INFORMATION FOR SEQ ID NO:42:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8                                                                  (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-210                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                       ThrAlaArgGlyTrpGluCysThr                                                       5                                                                              (2) INFORMATION FOR SEQ ID NO:43:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-212                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                       XaaAspSerProTrpThrXaaIleSerGlySer                                              510                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11                                                                 (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-214                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                       LeuArgSerCysGlyThrHisSerProTyrMet                                              510                                                                            (2) INFORMATION FOR SEQ ID NO:45:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8                                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-215/34A                                                      (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                       ThrTyrLeuHisThrTyrGluSer                                                       5                                                                              (2) INFORMATION FOR SEQ ID NO:46:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13                                                                 (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                       AlaIleIleAlaAsnLeuThrCysLysLysProAspGln                                        510                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8                                                                  (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-216                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                       IleValGlyGlnLeuMetAspGly                                                       5                                                                              (2) INFORMATION FOR SEQ ID NO:48:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9                                                                  (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-218/44                                                       (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                       ThrSerArgSerTyrProGluIleLeu                                                    5                                                                              (2) INFORMATION FOR SEQ ID NO:49:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9                                                                  (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-223B/24                                                      (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                       GlnAlaGluValSerSerValProAsp                                                    5                                                                              (2) INFORMATION FOR SEQ ID NO:50:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14                                                                 (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-224                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                       ArgCysPheGluLeuGlnGluAlaGlyProProAspAspCys                                     510                                                                            (2) INFORMATION FOR SEQ ID NO:51:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-229                                                          (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                       SerTyrThrSerCysCysHisAspPheAspGluLeu                                           510                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16                                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: ATX-224/53                                                       (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                       GlnMetSerTyrGlyPheLeuPheProProTyrLeu                                           1510                                                                           SerSerSerPro                                                                   15                                                                             (2) INFORMATION FOR SEQ ID NO:53:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 117                                                                (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: cDNA                                                          (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE:                                                               (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE: Liver                                                         (G) CELL TYPE:                                                                 (H) CELL LINE:                                                                 (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: 5'end of human liver                                    ATX gene                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                       ATGGCAAGGAGGAGCTCGTTCCAGTCGTGTCAAGATATAT40                                     CCCTGTTCACTTTTGCCGTTGGAGTCAATATCTGCTTAGG80                                     ATTCACTGCACATCGAATTAAGAGAGCAGAAGGATGG117                                       (2) INFORMATION FOR SEQ ID NO:54:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39                                                                 (B) TYPE: amino acids                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: Peptide                                                       (iii) HYPOTHETICAL: No                                                         (v) FRAGMENT TYPE: N-terminal fragment                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Human                                                            (B) STRAIN:                                                                    (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE: Liver                                                         (G) CELL TYPE:                                                                 (H) CELL LINE:                                                                 (I) ORGANELLE:                                                                 (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: N-terminal region                                       including transmembrane domain of liver                                        ATX protein                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                       MetAlaArgArgSerSerPheGlnSerCysGlnAsp                                           1510                                                                           IleSerLeuPheThrPheAlaValGlyValAsnIle                                           1520                                                                           CysLeuGlyPheThrAlaHisArgIleLysArgAla                                           253035                                                                         GluGlyTrp                                                                      (2) INFORMATION FOR SEQ ID NO:55:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: Yes                                                           (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Primer from 5'end of                                    4C11                                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                       GCTCAGATAAGGAGGAAAGAG21                                                        (2) INFORMATION FOR SEQ ID NO:56:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: Yes                                                           (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested primers from                                     4C11                                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                       GAATCCGTAGGACATCTGCTT21                                                        (2) INFORMATION FOR SEQ ID NO:57:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: Yes                                                           (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested primers from                                     4C11                                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                       TGTAGGCCAAACAGTTCTGAC21                                                        (2) INFORMATION FOR SEQ ID NO:58:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested sense primer                                     deduced from ATX-101, wherein N is                                             inosine                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                       AAYTCNATGCARACNGTNTTYGTNG25                                                    (2) INFORMATION FOR SEQ ID NO:59:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested primer of ATX                                    101, wherein N is inosine                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                       TTYGTNGGNTAYGGNCCNACNTTYAA26                                                   (2) INFORMATION FOR SEQ ID NO:60:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested primer deduced                                   from ATX- 103, wherein N is inosine                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                       AAYTAYCTNACNAAYGTNGAYGAYAT26                                                   (2) INFORMATION FOR SEQ ID NO:61:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested primer deduced                                   from ATX- 103, wherein N is inosine                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                       GAYGAYATNACNCTNGTNCCNGGNAC26                                                   (2) INFORMATION FOR SEQ ID NO:62:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Nested primer deduced                                   from ATX- 103, wherein N is inosine                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                       TGYTTYGARYTNCARGARGCNGGNCCNCC29                                                (2) INFORMATION FOR SEQ ID NO:63:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                       GCTGTCTTCAAACACAGC18                                                           (2) INFORMATION FOR SEQ ID NO:64:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                       CTGGTGGCTGTAATCCATAGC21                                                        (2) INFORMATION FOR SEQ ID NO:65:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21                                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (ix) FEATURE:                                                                  (A) NAME/KEY:                                                                  (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION: Primer for 5'end of                                     N-tera 2D1 sequence                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                       CGTGAAGGCAAAGAGAACACG21                                                        (2) INFORMATION FOR SEQ ID NO:66:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3104                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: N-tera 2D1 ATX cDNA                                              (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                       AGTGCACTCCGTGAAGGCAAAGAGAACACGCTGCAAAAGG40                                     CTTTCCAATAATCCTCGACATGGCAAGGAGGAGCTCGTTC80                                     CAGTCGTGTCAGATAATATCCCTGTTCACTTTTGCCGTTG120                                    GAGTCAATATCTGCTTAGGATTCACTGCACATCGAATTAA160                                    GAGAGCAGAAGGATGGGAGGAAGGTCCTCCTACAGTGCTA200                                    TCAGACTCCCCCTGGACCAACATCTCCGGATCTTGCAAGG240                                    GCAGGTGCTTTGAACTTCAAGAGGCTGGACCTCCTGATTG280                                    TCGCTGTGACAACTTGTGTAAGAGCTATACCAGTTGCTGC320                                    CATGACTTTGATGAGCTGTGTTTGAAGACAGCCCGTGCGT360                                    GGGAGTGTACTAAGGACAGATGTGGAGAAGTCAGAAATGA400                                    AGAAAATGCCTGTCACTGCTCAGAGGACTGCTTGGCCAGG440                                    GGAGACTGCTGTACCAATTACCAAGTGGTTTGCAAAGGAG480                                    AGTCGCATTGGGTTGATGATGACTGTGAGGAAATAAAGGC520                                    CGCAGAATGCCCTGCAGGGTTTGTTCGCCCTCCATTAATC560                                    ATCTTCTCCGTGGATGGCTTCCGTGCATCATACATGAAGA600                                    AAGGCAGCAAAGTCATGCCTAATATTGAAAAACTAAGGTC640                                    TTGTGGCACACACTCGCCCCACATGAGGCCGGTGTACCCA680                                    ACTAAAACCTTTCCTAACTTATACACTTTGGCCACTGGGC720                                    TATATCCAGAATCACATGGAATTGTTGGCAATTCAATGTA760                                    TGATCCTGTATTTGATGCCACTTTTCATCTGCGAGGGCGA800                                    GAGAAATTTAATCATAGATGGTGGGGAGGTCAACCGCTAT840                                    GGATTACAGCCACCAAGCAAAGGGGTGAAAGCTGGAACAT880                                    TCTTTTGGTCTGTTGTCATCCCTCACGAGCGGAGATATTA920                                    ACCATATTGCAGTGGCTCACCCTGCCAGATCATGAGAGGC960                                    TTCGGTCTATGCCTTCTATTCTGAGCAACCTGATTTCTCT1000                                   GGACACAAATATGCCTTTCGGCCCTGAGATGACAAATCCT1040                                   CTGAGGGAAATCGACAAAATTGTGGGGCAATTAATGGATG1080                                   GACTGAAACAACTAAAACTGCATCGGTGTGTCAACGTCAT1120                                   CTTTGTCGGAGACCATGGAATGGAAGATGTCACATGTGAT1160                                   AGAACTGAGTTCTTGAGTAATTACCTAACTAATGTGGATG1200                                   ATATTACTTTAGTGCCTGGAACTCTAGGAATTCGATCCAA1240                                   ATTTAGCAACAATGCTAAATATGACCCCAAAGCCATTATT1280                                   GCCAATCTCACGTGTAAAAAACCAGATCAGCACTTTAAGC1320                                   CTTACTTGAAACAGCACCTTCCCAAACGTTTGCACTATGC1360                                   CAACAACAGAAGAATTGAGGATATCCATTTATTGGTGGAA1400                                   CGCAGATGGCATGTTGCAAGGAAACCTTTGGATGTTTATA1440                                   AGAAACCATCAGGAAAATGCTTTTTCCAGGGAGACCACGG1480                                   ATTTGATAACAAGGTCAACAGCATGCAGACTGTTTTTGTA1520                                   GGTTATGGCCCAACATTTAAGTACAAGACTAAAGTGCCTC1560                                   CATTTGAAAACATTGAACTTTACAATGTTATGTGTGATCT1600                                   CCTGGGATTGAAGCCAGCTCCTAATAATGGGACCCATGGA1640                                   AGTTTGAATCATCTCCTGCGCACTAATACCTTCAGGCCAA1680                                   CCATGCCAGAGGAAGTTACCAGACCCAATTATCCAGGGAT1720                                   TATGTACCTTCAGTCTGATTTTGACCTGGGCTGCACTTGT1760                                   GATGATAAGGTAGAGCCAAAGAACAAGTTGGATGAACTCA1800                                   ACAAACGGCTTCATACAAAAGGGTCTACAGAAGAGAGACA1840                                   CCTCCTCTATGGGCGACCTGCAGTGCTTTATCGGACTAGA1880                                   TATGATGTCTTATATCACACTGACTTTGAAAGTGGTTATA1920                                   GTGAAATATTCCTAATGCCACTCTGGACATCATATACTGT1960                                   TTCCAAACAGGCTGAGGTTTCCAGCGTTCCTGACCATCTG2000                                   ACCAGTTGCGTCCGGCCTGATGTCCGTGTTTCTCCGAGTT2040                                   TCAGTCAGAACTGTTTGGCCTACAAAAATGATAAGCAGAT2080                                   GTCCTACGGATTCCTCTTTCCTCCTTATCTGAGCTCTTCA2120                                   CCAGAGGCTAAATATGATGCATTCCTTGTAACCAATATGG2160                                   TTCCAATGTATCCTGCTTTCAAACGGGTCTGGAATTATTT2200                                   CCAAAGGGTATTGGTGAAGAAATATGCTTCGGAAAGAAAT2240                                   GGAGTTAACGTGATAAGTGGACCAATCTTCGACTATGACT2280                                   ATGATGGCTTACATGACACAGAAGACAAAATAAAACAGTA2320                                   CGTGGAAGGCAGTTCCATTCCTGTTCCAACTCACTACTAC2360                                   AGCATCATCACCAGCTGTCTGGATTTCACTCAGCCTGCCG2400                                   ACAAGTGTGACGGCCCTCTCTCTGTGTCCTCCTTCATCCT2440                                   CCGTCACCGGCCTGACAACGAGGAGAGCTGCAATAGCTCA2480                                   GAGGACGAATCAAAATGGGTAGAAGAACTCATGAAGATGC2520                                   ACACGGCTAGGGTGCGTGACATTGAACATCTCACCAGCCT2560                                   GGACTTCTTCCGAAAGACCAGCCGCAGCTACCCAGAAATC2600                                   CTGACACTCAAGACATACCTGCATACATATGAGAGCGAGA2640                                   TTTAACTTTCTGAGCATCTGCAGTACAGTCTTATCAACTG2680                                   GTTGTATATTTTTATATTGTTTTTGTATTTATTAATTTGA2720                                   AACCAGGACATTAAAAATGTTAGTATTTTAATCCTGTACC2760                                   AAATCTGACATATTATGCCTGAATGACTCCACTGTTTTTC2800                                   TCTAATGCTTGATTTAGGTAGCCTTGTGTTCTGAGTAGAG2840                                   CTTGTAATAAATACTGCAGCTTGAGTTTTTAGTGGAAGCT2880                                   TCTAAATGGTGCTGCAGATTTGATATTTGCATTGAGGAAA2920                                   TATTAATTTTCCAATGCACAGTTGCCACATTTAGTCCTGT2960                                   ACTGTATGGAAACACTGATTTTGTAAAGTTGCCTTTATTT3000                                   GCTGTTAACTGTTAACTATGACAGATATATTTAAGCCTTA3040                                   TAAACCAATCTTAAACATAATAAATCACACATTCAGTTTT3080                                   TTCTGGTAAAAAAAAAAAAAAAAA3104                                                   (2) INFORMATION FOR SEQ ID NO:67:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 861                                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: N-tera 2D1 ATX protein                                           (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                       MetAlaArgArgSerSerPheGlnSerCysGlnIleIleSerLeuPhe                               151015                                                                         ThrPheAlaValGlyValAsnIleCysLeuGlyPheThrAlaHisArg                               202530                                                                         IleLysArgAlaGluGlyTrpGluGluGlyProProThrValLeuSer                               354045                                                                         AspSerProTrpThrAsnIleSerGlySerCysLysGlyArgCysPhe                               505560                                                                         GluLeuGlnGluAlaGlyProProAspCysArgCysAspAsnLeuCys                               65707580                                                                       LysSerTyrThrSerCysCysHisAspPheAspGluLeuCysLeuLys                               859095                                                                         ThrAlaArgAlaTrpGluCysThrLysAspArgCysGlyGluValArg                               100105110                                                                      AsnGluGluAsnAlaCysHisCysSerGluAspCysLeuAlaArgGly                               115120125                                                                      AspCysCysThrAsnTyrGlnValValCysLysGlyGluSerHisTrp                               130135140                                                                      ValAspAspAspCysGluGluIleLysAlaAlaGluCysProAlaGly                               145150155160                                                                   PheValArgProProLeuIleIlePheSerValAspGlyPheArgAla                               165170175                                                                      SerTyrMetLysLysGlySerLysValMetProAsnIleGluLysLeu                               180185190                                                                      ArgSerCysGlyThrHisSerProHisMetArgProValTyrProThr                               195200205                                                                      LysThrPheProAsnLeuTyrThrLeuAlaThrGlyLeuTyrProGlu                               210215220                                                                      SerHisGlyIleValGlyAsnSerMetTyrAspProValPheAspAla                               225230235240                                                                   ThrPheHisLeuArgGlyArgGluLysPheAsnHisArgTrpTrpGly                               245250255                                                                      GlyGlnProLeuTrpIleThrAlaThrLysGlnArgGlyGluSerTrp                               260265270                                                                      AsnIleLeuLeuValCysCysHisProSerArgAlaGluIleLeuThr                               275280285                                                                      IleLeuGlnTrpLeuThrLeuProAspHisGluArgLeuArgSerMet                               290295300                                                                      ProSerIleLeuSerAsnLeuIleSerLeuAspThrAsnMetProPhe                               305310315320                                                                   GlyProGluMetThrAsnProLeuArgGluIleAspLysIleValGly                               325330335                                                                      GlnLeuMetAspGlyLeuLysGlnLeuLysLeuHisArgCysValAsn                               340345350                                                                      ValIlePheValGlyAspHisGlyMetGluAspValThrCysAspArg                               355360365                                                                      ThrGluPheLeuSerAsnTyrLeuThrAsnValAspAspIleThrLeu                               370375380                                                                      ValProGlyThrLeuGlyIleArgSerLysPheSerAsnAsnAlaLys                               385390395400                                                                   TyrAspProLysAlaIleIleAlaAsnLeuThrCysLysLysProAsp                               405410415                                                                      GlnHisPheLysProTyrLeuLysGlnHisLeuProLysArgLeuHis                               420425430                                                                      TyrAlaAsnAsnArgArgIleGluAspIleHisLeuLeuValGluArg                               435440445                                                                      ArgTrpHisValAlaArgLysProLeuAspValTyrLysLysProSer                               450455460                                                                      GlyLysCysPhePheGlnGlyAspHisGlyPheAspAsnLysValAsn                               465470475480                                                                   SerMetGlnThrValPheValGlyTyrGlyProThrPheLysTyrLys                               485490495                                                                      ThrLysValProProPheGluAsnIleGluLeuTyrAsnValMetCys                               500505510                                                                      AspLeuLeuGlyLeuLysProAlaProAsnAsnGlyThrHisGlySer                               515520525                                                                      LeuAsnHisLeuLeuArgThrAsnThrPheArgProThrMetProGlu                               530535540                                                                      GluValThrArgProAsnTyrProGlyIleMetTyrLeuGlnSerAsp                               445450555560                                                                   PheAspLeuGlyCysThrCysAspAspLysValGluProLysAsnLys                               565570575                                                                      LeuAspGluLeuAsnLysArgLeuHisThrLysGlySerThrGluGlu                               580585590                                                                      ArgHisLeuLeuTyrGlyArgProAlaValLeuTyrArgThrArgTyr                               595600605                                                                      AspValLeuTyrHisThrAspPheGluSerGlyTyrSerGluIlePhe                               610615620                                                                      LeuMetProLeuTrpThrSerTyrThrValSerLysGlnAlaGluVal                               625630635640                                                                   SerSerValProAspHisLeuThrSerCysValArgProAspValArg                               645650655                                                                      ValSerProSerPheSerGlnAsnCysLeuAlaTyrLysAsnAspLys                               660665670                                                                      GlnMetSerTyrGlyPheLeuPheProProTyrLeuSerSerSerPro                               675680685                                                                      GluAlaLysTyrAspAlaPheLeuValThrAsnMetValProMetTyr                               690695700                                                                      ProAlaPheLysArgValTrpAsnTyrPheGlnArgValLeuValLys                               705710715720                                                                   LysTyrAlaSerGluArgAsnGlyValAsnValIleSerGlyProIle                               725730735                                                                      PheAspTyrAspTyrAspGlyLeuHisAspThrGluAspLysIleLys                               740745750                                                                      GlnTyrValGluGlySerSerIleProValProThrHisTyrTyrSer                               755760765                                                                      IleIleThrSerCysLeuAspPheThrGlnProAlaAspLysCysAsp                               770775780                                                                      GlyProLeuSerValSerSerPheIleLeuArgHisArgProAspAsn                               785790795800                                                                   GluGluSerCysAsnSerSerGluAspGluSerLysTrpValGluGlu                               805810815                                                                      LeuMetLysMetHisThrAlaArgValArgAspIleGluHisLeuThr                               820825830                                                                      SerLeuAspPhePheArgLysThrSerArgSerTyrProGluIleLeu                               835840845                                                                      ThrLeuLysThrTyrLeuHisThrTyrGluSerGluIle                                        850855860                                                                      (2) INFORMATION FOR SEQ ID NO:68:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3251                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: A2058 ATX cDNA                                                   (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                       CGTGAAGGCAAAGAGAACACGCTGCAAAAGGCTTCCAAGA40                                     ATCCTCGACATGGCAAGGAGGAGCTCGTTCCAGTCGTGTC80                                     AGATAATATCCCTGTTCACTTTTGCCGTTGGAGTCAGTAT120                                    CTGCTTAGGATTCACTGCACATCGAATTAAGAGAGCAGAA160                                    GGATGGGAGGAAGGTCCTCCTACAGTGCTATCAGACTCCC200                                    CCTGGACCAACATCTCCGGATCTTGCAAGGGCAGGTGCTT240                                    TGAACTTCAAGAGGCTGGACCTCCTGATTGTCGCTGTGAC280                                    AACTTGTGTAAGAGCTATACCAGTTGCTGCCATGACTTTG320                                    ATGAGCTGTGTTTGAAGACAGCCCGTGGCTGGGAGTGTAC360                                    TAAGGACAGATGTGGAGAAGTCAGAAATGAAGAAAATGCC400                                    TGTCACTGCTCAGAGGACTGCTTGGCCAGGGGAGACTGCT440                                    GTACCAATTACCAAGTGGTTTGCAAAGGAGAGTCGCATTG480                                    GGTTGATGATGACTGTGAGGAAATAAAGGCCGCAGAATGC520                                    CCTGCAGGGTTTGTTCGCCCTCCATTAATCATCTTCTCCG560                                    TGGATGGCTTCCGTGCATCATACATGAAGAAAGGCAGCAA600                                    AGTCATGCCTAATATTGAAAAACTAAGGTCTTGTGGCACA640                                    CACTCTCCCTACATGAGGCCGGTGTACCCAACTAAAACCT680                                    TTCCTAACTTATACACTTTGGCCACTGGGCTATATCCAGA720                                    ATCACATGGAATTGTTGGCAATTCAATGTATGATCCTGTA760                                    TTTGATGCCACTTTTCATCTGCGAGGGCGAGAGAAATTTA800                                    ATCATAGATGGTGGGGAGGTCAACCGCTATGGATTACAGC840                                    CACCAAGCAAGGGGTGAAAGCTGGAACATTCTTTTGGTCT880                                    GTTGTCATCCCTCACGAGCGGAGAATATTAACCATATTGC920                                    GGTGGCTCACCCTGCCAGATCATGAGAGGCCTTCGGTCTA960                                    TGCCTTCTATTCTGAGCAACCTGATTTCTCTGGACACAAA1000                                   TATGGCCCTTTCGGCCCTGAGGAGAGTAGTTATGGCTCAC1040                                   CTTTTACTCCGGCTAAGAGACCTAAGAGGAAAGTTGCCCC1080                                   TAAGAGGAGACAGGAAAGACCAGTTGCTCCTCCAAAGAAA1120                                   AGAAGAAGAAAAATACATAGGATGGATCATTATGCTGCGG1160                                   AAACTCGTCAGGACAAAATGACAAATCCTCTGAGGGAAAT1200                                   CGACAAAATTGTGGGGCAATTAATGGATGGACTGAAACAA1240                                   CTAAAACTGCGTCGGTGTGTCAACGTCATCTTTGTCGGAG1280                                   ACCATGGAATGGAAGATGTCACATGTGATAGAACTGAGTT1320                                   CTTGAGTAATTACCTAACTAATGTGGATGATATTACTTTA1360                                   GTGCCTGGAACTCTAGGAAGAATTCGATCCAAATTTAGCA1400                                   ACAATGCTAAATATGACCCCAAAGCCATTATTGCCAATCT1440                                   CACGTGTAAAAAACCAGATCAGCACTTTAAGCCTTACTTG1480                                   AAACAGCACCTTCCCAAACGTTTGCACTATGCCAACAACA1520                                   GAAGAATTGAGGATATCCATTTATTGGTGGAACGCAGATG1560                                   GCATGTTGCAAGGAAACCTTTGGATGTTTATAAGAAACCA1600                                   TCAGGAAAATGCTTTTTCCAGGGAGACCACGGATTTGATA1640                                   ACAAGGTCAACAGCATGCAGACTGTTTTTGTAGGTTATGG1680                                   CCCAACATTTAAGTACAAGACTAAAGTGCCTCCATTTGAA1720                                   AACATTGAACTTTACAATGTTATGTGTGATCTCCTGGGAT1760                                   TGAAGCCAGCTCCTAATAATGGGACCCATGGAAGTTTGAA1800                                   TCATCTCCTGCGCACTAATACCTTCAGGCCAACCATGCCA1840                                   GAGGAAGTTACCAGACCCAATTATCCAGGGATTATGTACC1880                                   TTCAGTCTGATTTTGACCTGGGCTGCACTTGTGATGATAA1920                                   GGTAGAGCCAAAGAACAAGTTGGATGAACTCAACAAACGG1960                                   CTTCATACAAAAGGGTCTACAGAAGAGAGACACCTCCTCT2000                                   ATGGGCGACCTGCAGTGCTTTATCGGACTAGATATGATAT2040                                   CTTATATCACACTGACTTTGAAAGTGGTTATAGTGAAATA2080                                   TTCCTAATGCTACTCTGGACATCATATACTGTTTCCAAAC2120                                   AGGCTGAGGTTTCCAGCGTTCCTGACCATCTGACCAGTTG2160                                   CGTCCGGCCTGATGTCCGTGTTTCTCCGAGTTTCAGTCAG2200                                   AACTGTTTGGCCTACAAAAATGATAAGCAGATGTCCTACG2240                                   GATTCCTCTTTCCTCCTTATCTGAGCTCTTCACCAGAGGC2280                                   TAAATATGATGCATTCCTTGTAACCAATATGGTTCCAATG2320                                   TATCCTGCTTTCAAACGGGTCTGGAATTATTTCCAAAGGG2360                                   TATTGGTGAAGAAATATGCTTCGGAAAGAAATGGAGTTAA2400                                   CGTGATAAGTGGACCAATCTTCGACTATGACTATGATGGC2440                                   TTACATGACACAGAAGACAAAATAAAACAGTACGTGGAAG2480                                   GCAGTTCCATTCCTGTTCCAACTCACTACTACAGCATCAT2520                                   CACCAGCTGTCTGGATTTCACTCAGCCTGCCGACAAGTGT2560                                   GACGGCCCTCTCTCTGTGTCCTCCTTCATCCTGCCTCACC2600                                   GGCCTGACAACGAGGAGAGCTGCAATAGCTCAGAGGACGA2640                                   ATCAAAATGGGTAGAAGAACTCATGAAGATGCACACAGCT2680                                   AGGGTGCGTGACATTGAACATCTCACCAGCCTGGACTTCT2720                                   TCCGAAAGACCAGCCGCAGCTACCCAGAAATCCTGACACT2760                                   CAAGACATACCTGCATACATATGAGAGCGAGATTTAACTT2800                                   TCTGAGCATCTGCAGTACAGTCTTATCAACTGGTTGTATA2840                                   TTTTTATATTGTTTTTGTATTTATTAATTTGAAACCAGGA2880                                   CATTAAAAATGTTAGTATTTTAATCCTGTACCAAATCTGA2920                                   CATATTATGCCTGAATGACTCCACTGTTTTTCTCTAATGC2960                                   TTGATTTAGGTAGCCTTGTGTTCTGAGTAGAGCTTGTAAT3000                                   AAATACTGCAGCTTGAGAAAAAGTGGAAGCTTCTAAATGG3040                                   TGCTGCAGATTTGATATTTGCATTGAGGAAATATTAATTT3080                                   TCCAATGCACAGTTGCCACATTTAGTCCTGTACTGTATGG3120                                   AAACACTGATTTTGTAAAGTTGCCTTTATTTGCTGTTAAC3160                                   TGTTAACTATGACAGATATATTTAAGCCTTATAAACCAAT3200                                   CTTAAACATAATAAATCACACATTCAGTTTTAAAAAAAAA3240                                   AAAAAAAAAAA3251                                                                (2) INFORMATION FOR SEQ ID NO:69:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 915                                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: Unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: No                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: A2058 ATX protein                                                (B) LOCATION:                                                                  (C) IDENTIFICATION METHOD:                                                     (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                       MetAlaArgArgSerSerPheGlnSerCysGlnIle                                           1510                                                                           IleSerLeuPheThrPheAlaValGlyValSerIle                                           1520                                                                           CysLeuGlyPheThrAlaHisArgIleLysArgAla                                           253035                                                                         GluGlyTrpGluGluGlyProProThrValLeuSer                                           4045                                                                           AspSerProTrpThrAsnIleSerGlySerCysLys                                           505560                                                                         GlyArgCysPheGluLeuGlnGluAlaGlyProPro                                           6570                                                                           AspCysArgCysAspAsnLeuCysLysSerTyrThr                                           7580                                                                           SerCysCysHisAspPheAspGluLeuCysLeuLys                                           859095                                                                         ThrAlaArgGlyTrpGluCysThrLysAspArgCys                                           100105                                                                         GlyGluValArgAsnGluGluAsnAlaCysHisCys                                           110115120                                                                      SerGluAspCysLeuAlaArgGlyAspCysCysThr                                           125130                                                                         AsnTyrGlnValValCysLysGlyGluSerHisTrp                                           135140                                                                         ValAspAspAspCysGluGluIleLysAlaAlaGlu                                           145150155                                                                      CysProAlaGlyPheValArgProProLeuIleIle                                           160165                                                                         PheSerValAspGlyPheArgAlaSerTyrMetLys                                           170175180                                                                      LysGlySerLysValMetProAsnIleGluLysLeu                                           185190                                                                         ArgSerCysGlyThrHisSerProTyrMetArgPro                                           195200                                                                         ValTyrProThrLysThrPheProAsnLeuTyrThr                                           205210215                                                                      LeuAlaThrGlyLeuTyrProGluSerHisGlyIle                                           220225                                                                         ValGlyAsnSerMetTyrAspProValPheAspAla                                           230235240                                                                      ThrPheHisLeuArgGlyArgGluLysPheAsnHis                                           245250                                                                         ArgTrpTrpGlyGlyGlnProLeuTrpIleThrAla                                           255260                                                                         ThrLysGlnGlyValLysAlaGlyThrPhePheTrp                                           265270275                                                                      SerValValIleProHisGluArgArgIleLeuThr                                           280285                                                                         IleLeuArgTrpLeuThrLeuProAspHisGluArg                                           290295300                                                                      ProSerValTyrAlaPheTyrSerGluGlnProAsp                                           305310                                                                         PheSerGlyHisLysTyrGlyProPheGlyProGlu                                           315320                                                                         GluSerSerTyrGlySerProPheThrProAlaLys                                           325330335                                                                      ArgProLysArgLysValAlaProLysArgArgGln                                           340345                                                                         GluArgProValAlaProProLysLysArgArgArg                                           350355360                                                                      LysIleHisArgMetAspHisTyrAlaAlaGluThr                                           365370                                                                         ArgGlnAspLysMetThrAsnProLeuArgGluIle                                           375380                                                                         AspLysIleValGlyGlnLeuMetAspGlyLeuLys                                           385390395                                                                      GlnLeuLysLeuArgArgCysValAsnValIlePhe                                           400405                                                                         ValGlyAspHisGlyMetGluAspValThrCysAsp                                           410415420                                                                      ArgThrGluPheLeuSerAsnTyrLeuThrAsnVal                                           425430                                                                         AspAspIleThrLeuValProGlyThrLeuGlyArg                                           435440                                                                         IleArgSerLysPheSerAsnAsnAlaLysTyrAsp                                           445450455                                                                      ProLysAlaIleIleAlaAsnLeuThrCysLysLys                                           460465                                                                         ProAspGlnHisPheLysProTyrLeuLysGlnHis                                           470475480                                                                      LeuProLysArgLeuHisTyrAlaAsnAsnArgArg                                           485490                                                                         IleGluAspIleHisLeuLeuValGluArgArgTrp                                           495500                                                                         HisValAlaArgLysProLeuAspValTyrLysLys                                           505510515                                                                      ProSerGlyLysCysPhePheGlnGlyAspHisGly                                           520525                                                                         PheAspAsnLysValAsnSerMetGlnThrValPhe                                           530535540                                                                      ValGlyTyrGlyProThrPheLysTyrLysThrLys                                           545550                                                                         ValProProPheGluAsnIleGluLeuTyrAsnVal                                           555560                                                                         MetCysAspLeuLeuGlyLeuLysProAlaProAsn                                           565570575                                                                      AsnGlyThrHisGlySerLeuAsnHisLeuLeuArg                                           580585                                                                         ThrAsnThrPheArgProThrMetProGluGluVal                                           590595600                                                                      ThrArgProAsnTyrProGlyIleMetTyrLeuGln                                           605610                                                                         SerAspPheAspLeuGlyCysThrCysAspAspLys                                           615620                                                                         ValGluProLysAsnLysLeuAspGluLeuAsnLys                                           625630635                                                                      ArgLeuHisThrLysGlySerThrGluGluArgHis                                           640645                                                                         LeuLeuTyrGlyArgProAlaValLeuTyrArgThr                                           650655660                                                                      ArgTyrAspIleLeuTyrHisThrAspPheGluSer                                           665670                                                                         GlyTyrSerGluIlePheLeuMetLeuLeuTrpThr                                           675680                                                                         SerTyrThrValSerLysGlnAlaGluValSerSer                                           685690695                                                                      ValProAspHisLeuThrSerCysValArgProAsp                                           700705                                                                         ValArgValSerProSerPheSerGlnAsnCysLeu                                           710715720                                                                      AlaTyrLysAsnAspLysGlnMetSerTyrGlyPhe                                           725730                                                                         LeuPheProProTyrLeuSerSerSerProGluAla                                           735740                                                                         LysTyrAspAlaPheLeuValThrAsnMetValPro                                           745750755                                                                      MetTyrProAlaPheLysArgValTrpAsnTyrPhe                                           760765                                                                         GlnArgValLeuValLysLysTyrAlaSerGluArg                                           770775780                                                                      AsnGlyValAsnValIleSerGlyProIlePheAsp                                           785790                                                                         TyrAspTyrAspGlyLeuHisAspThrGluAspLys                                           795800                                                                         IleLysGlnTyrValGluGlySerSerIleProVal                                           805810815                                                                      ProThrHisTyrTyrSerIleIleThrSerCysLeu                                           820825                                                                         AspPheThrGlnProAlaAspLysCysAspGlyPro                                           830835840                                                                      LeuSerValSerSerPheIleLeuProHisArgPro                                           845850                                                                         AspAsnGluGluSerCysAsnSerSerGluAspGlu                                           855860                                                                         SerLysTrpValGluGluLeuMetLysMetHisThr                                           865870875                                                                      AlaArgValArgAspIleGluHisLeuThrSerLeu                                           880885                                                                         AspPhePheArgLysThrSerArgSerTyrProGlu                                           890895900                                                                      IleLeuThrLeuLysThrTyrLeuHisThrTyr                                              905910                                                                         GluSerGluIle                                                                   916                                                                            __________________________________________________________________________ 

We claim:
 1. A DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to human autotaxin, or a fragment thereof having immunogenic activity or a biological activity associated with autotaxin having at least 10 amino acids.
 2. The DNA segment according to claim 1, wherein said DNA segment encodes the amino acid sequence selected from the group consisting of SEQ ID NO:5 through SEQ ID NO:11, SEQ ID NO:29 through. SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50 through SEQ ID NO:52 and SEQ ID NO:54, SEQ ID NO:67 and SEQ ID NO:69.
 3. A recombinant DNA molecule comprising a vector and the DNA segment according to claim
 1. 4. A cell that contains the recombinant DNA molecule according to claim
 3. 5. A method of producing a recombinant autotaxin polypeptide said method comprising:culturing a cell containing the recombinant DNA molecule of claim 3 under conditions such that the DNA segment is expressed, producing said polypeptide; and isolating said polypeptide.
 6. An isolated DNA encoding a human autotaxin protein or fragment thereof having immunogenic activity or a biological activity associated with autotaxin wherein said DNA includes a nucleic acid sequence selected from the group consisting of SEQ ID NO:35 and SEQ ID NO:37.
 7. The DNA segment according to claim 1, wherein said DNA fragment comprises any one of the SEQ ID NO:12 through SEQ ID NO:25, or SEQ ID NO:39 through SEQ ID NO:52.
 8. The DNA segment according to claim 6 wherein said DNA segment comprises any one of the SEQ ID NO:12 through SEQ ID NO:25.
 9. The DNA segment according to claim 1, wherein said DNA segment encodes an amino acid sequence comprising a phosphodiesterase binding site. 